6-6 Fused Bicyclic Heteroaryl Compounds and their Use as LATS Inhibitors

ABSTRACT

The present invention is related to 6-6 Fused Bicyclic Heteroaryl Compounds of the Formula A2 or A1 and their Use as LATS Inhibitors, or a salt, stereoisomer or pharmaceutical composition thereof; wherein the variables are as defined herein. 
     
       
         
         
             
             
         
       
     
     The present invention further relates to a method of LATS inhibition in a cell population using a compound of Formula A1, or a salt, stereoisomer or pharmaceutical composition thereof. The present invention further provides a method for manufacturing compounds of the invention, and its therapeutic uses. The invention further provides methods to their preparation, to their medical use, their use in the treatment and management of diseases or disorders.

The present application is a continuation of U.S. patent applicationSer. No. 15/963,816, filed on Apr. 26, 2018; which claims priority toU.S. Provisional Application Ser. No. 62/491,475, filed on Apr. 28,2017, and to U.S. Provisional Application Ser. No. 62/491,484, filed onApr. 28, 2017, and to U.S. Provisional Application Ser. No. 62/491,573,filed on Apr. 28, 2017, and to U.S. Provisional Application Ser. No.62/491,526, filed on Apr. 28, 2017, and to U.S. Provisional ApplicationSer. No. 62/650,232, filed on Mar. 29, 2018, the disclosures of whichare entirely and specifically incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Feb. 14, 2023, isnamed 14452_0043-01000_SL.xml and is 28,830 bytes in size.

INTRODUCTION

The present invention relates to LATS (large tumor suppressor kinase)inhibitors. The present invention further relates to 6-6 fused bicyclicheteroaryl compounds and compositions comprising such compounds.

The present invention also relates to ex-vivo use of such compounds toproduce cellular material for cell therapy/transplantation. The presentinvention further relates to methods of generating an expandedpopulation of cells, such as an expanded population of ocular cells forexample comprising limbal stem cells (LSCs) or corneal endothelial cells(CECs) involving the use of a LATS inhibitor, as well as the populationof cells such as ocular cells comprising for example limbal stem cells(LSCs) or corneal endothelial cells (CECs) and preparations, uses andmethods of therapy comprising said cells.

The present invention also relates to 6-6 fused bicyclic heteroarylcompounds, compositions comprising such compounds, and their use in inpromoting wound healing, particularly for treatment of burns, acute andchronic skin ulcers, including vascular, diabetic and pressure ulcers,such as venous leg ulcers, diabetic foot ulcers, pressure ulcers.

The present invention additionally relates to 6-6 fused bicyclicheteroaryl compounds, compositions comprising such compounds, and theiruse in liver regeneration and liver regrowth as well as in theprevention of damage and in the maintenance or improvement of functionof organs ex-vivo, with or without perfusion devices.

BACKGROUND OF THE INVENTION

Organ regeneration and/or healing is an issue crucial to treat manyserious health issues.

For example in the eye, it is known that corneal blindness is the thirdleading cause of blindness worldwide. Approximately half of all thecornea transplants worldwide are performed for treatment of cornealendothelial dysfunction.

The cornea is a transparent tissue comprising different layers: cornealepithelium, Bowman's membrane, stroma, Descemet's Membrane andendothelium. The corneal endothelium also comprises a monolayer of humancorneal endothelial cells and helps maintain corneal transparency viaits barrier and ionic pump functions. It plays a crucial role inmaintaining the balance of fluid, nutrients and salts between thecorneal stroma and the aqueous humor. To maintain transparency,endothelial cell density must be maintained, however endothelial celldensity can be significantly decreased as a result of trauma, disease orendothelial dystrophies. The density of the cells also decreases withaging. Human corneal endothelium has a limited propensity to proliferatein vivo. If the density of cells falls too low, the barrier function maybe compromised. Loss of endothelial barrier function results in cornealedema and loss of visual acuity. The clinical condition of bullouskeratopathy may be one resulting complication.

Currently the only treatment for blindness caused by corneal endothelialdysfunction is corneal transplantation. Although corneal transplantationis one of the most common forms of organ transplantation, theavailability of donor corneas required is extremely limited. A 2012-2013global survey quantified the considerable shortage of corneal grafttissue, finding that only one cornea is available for every 70 needed(Gain at el., (2016) Global Survey of Corneal Transplantation and EyeBanking. JAMA Ophthalmol. 134:167-173).

New therapeutic approaches to supply corneal endothelial cells for thetreatment of corneal endothelial dysfunction are thus greatly needed.

The corneal epithelium also needs to be maintained in the eye. Thecorneal epithelium is composed of a layer of basal cells and multiplelayers of a non-keratinized, stratified, squamous epithelium. It isessential in maintaining the clarity and the regular refractive surfaceof the cornea. It acts as a transparent, renewable protective layer overthe corneal stroma and is replenished by a stem cell population locatedin the limbus. In limbal stem cell deficiency, a condition in whichlimbal stem cells are diseased or absent, a decrease in the number ofhealthy limbal stem cells results in a decreased capacity for cornealepithelium renewal.

Limbal stem cell deficiency may arise as a result of injuries fromchemical or thermal burns, ultraviolet and ionizing radiation, or evenas a result of contact lens wear; genetic disorders like aniridia, andimmune disorders such as Stevens Johnson syndrome and ocular cicatricialpemphigoid. Loss of limbal stem cells can be partial or total; and maybe unilateral or bilateral. Symptoms of limbal stem cell deficiencyinclude pain, photophobia, non healing painful corneal epithelialdefects, corneal neovascularization, replacement of the cornealepithelium by conjunctival epithelium, loss of corneal transparency anddecreased vision that can eventually lead to blindness.

A product for use in treating limbal stem cell deficiency was granted aconditional marketing authorisation in the European Union in 2015 (underthe name Holoclar®), making it the first Advanced Therapy MedicinalProduct (ATMP) containing stem cells in Europe. Holoclar is an ex vivoexpanded preparation of autologous human corneal epithelial cellscontaining stem cells. A biopsy of healthy limbal tissue is taken fromthe patient, expanded ex vivo and frozen until surgery. Foradministration to the patient the thawed cells are grown on a membranecomprising fibrin, and then surgically implanted onto the eye of thepatient. The therapy is intended for use in adults with moderate tosevere limbal stem cell deficiency due to physical or chemical ocularburns. (Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, PellegriniG. (2010) Limbal stem-cell therapy and long-term corneal regeneration. NEngl J Med. 363:147-155). However the method is limited in that it isfor autologous use only and there must be enough surviving limbus in oneeye to allow a minimum of 1-2 square millimeters of undamaged tissue tobe extracted from the patient. There is also the risk that for eachspecific patient the culture of his/her cells may not be successful andthe patient cannot receive this treatment. Furthermore also feeder cellsof murine origin are used to prepare the Holoclar cell preparation,which introduces potential safety concerns, due to the risk of diseasetransmission and potential immunogenicity into the preparation for usein humans. Moreover the Holoclar cell preparation only containsapproximately 5% of limbal stem cells, as identified by p63alphastaining.

New therapeutic approaches to supply limbal stem cells for the treatmentof limbal stem cell deficiency are thus greatly needed.

Functional liver regeneration during homeostasis and in diseaseconditions is critical to maintaining essential physiological processes.Despite the liver's marked potential to regenerate, this process can beimpaired following severe acute or chronic liver injury. Liver damageand impaired liver regeneration often result in serious morbidity andmortality and therefore require life-saving liver transplantation.Unfortunately, the need for liver transplants currently far eclipses thesupply of available donor organs. As a result, many patients continue todie while awaiting a life-saving transplant. The use of split-livertransplants from deceased donors or partial-liver transplants fromliving donors is limited by graft size constraints. Transplantation of apartial liver that has an inadequate graft-to-recipient weight ratio(GRWR) increases the incidence of graft dysfunction and failure.Therapies that increase liver regrowth may allow transplantation ofpartial livers that otherwise would be deemed inadequate fortransplantation based on size. Alternatively, regenerating livers byinhibiting liver cell death, improving liver function and repairing theaberrant liver architecture could normalize liver function, preventingthe need for transplantation (Forbes S J and Newsome P N (2016) Liverregeneration—mechanisms and models to clinical application. NatureReviews Gastroenterology & Hepatology, 13(8):473-485; Dutkowski P,Linecker M, DeOliveira M L, MQllhaupt B, Clavien P A (2015) Challengesto Liver Transplantation and Strategies to Improve Outcomes.Gastroenterology, 148(2):307-323).

Hence, there remains an urgent need for more efficacious therapeutics topromote liver regrowth.

Chronic skin ulcers, including vascular, diabetic and pressure ulcers,constitute a major public health issue. The increased demand for woundcare is reflected in the association of wounds with comorbidities,increased mortality and patient's quality of life (Demidova-Rice T N,Hamblin M R, & Herman I M (2012) Acute and impaired wound healing:pathophysiology and current methods for drug delivery, part 1: normaland chronic wounds: biology, causes, and approaches to care. Advances inskin & wound care 25(7):304-314; Demidova-Rice T N, Hamblin M R, &Herman I M (2012) Acute and impaired wound healing: pathophysiology andcurrent methods for drug delivery, part 2: role of growth factors innormal and pathological wound healing: therapeutic potential and methodsof delivery. Advances in skin & wound care 25(8):349-370). The healthcare cost of patients with chronic wounds is around 25 billion dollarsper year in the US alone. No new chemical entities have been approved bythe FDA since the approval of Regranex (PDGF) in 1997, which has limitedefficacy (Eaglstein W H, Kirsner R S, & Robson M C (2012) Food and DrugAdministration (FDA) drug approval end points for chronic cutaneousulcer studies. Wound repair and regeneration: official publication ofthe Wound Healing Society [and] the European Tissue Repair Society20(6):793-796). Intrinsically, growth factors are not stable in theproteolytic environment of wound bed. Growth factor therapy could alsosuffer from low expression of its corresponding receptors in the wounds,as demonstrated in the clinic patient samples (Demidova-Rice T N,Hamblin M R, & Herman I M (2012) Acute and impaired wound healing:pathophysiology and current methods for drug delivery, part 2: role ofgrowth factors in normal and pathological wound healing: therapeuticpotential and methods of delivery. Advances in skin & wound care25(8):349-370).

Hence, there remains an urgent need for more efficacious therapeutics topromote wound healing in patients with chronic wounds.

New therapeutic approaches to promote cell proliferation are thusgreatly needed for conditions affecting a range of organs throughout thebody, such as the eye, liver and skin.

SUMMARY OF THE INVENTION

The present invention relates to compounds, salts thereof, andcompositions thereof, wherein the compounds are LATS (large tumorsuppressor kinase) inhibitors. These compounds have use in therapies forthe conditions and purposes detailed above.

Various aspects of the invention are described herein.

The present invention relates to a compound of Formula A2 or asubformulae thereof, or a salt, or stereoisomer thereof,

wherein X¹, ring A, R¹, R², R³, and R⁵ are as defined in the detaileddescription infra.

In a preferred embodiment, the compound is according to Formula I orFormula II, or subformulae thereof, or a salt thereof:

wherein ring A, R¹, R², R³, and R⁵ are as defined in the detaileddescription infra.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a therapeutically effective amount of a compound according tothe definition of Formula A2 or subformulae thereof, or apharmaceutically acceptable salt thereof, or subformulae thereof and oneor more pharmaceutically acceptable carriers.

In another aspect, the invention relates to a combination, in particulara pharmaceutical combination, comprising a therapeutically effectiveamount of the compound according to the definition of Formula A2 orsubformulae thereof, or a pharmaceutically acceptable salt thereof, andone or more therapeutically active agent.

In another aspect, the invention relates to compounds and compositionsthat may be used in therapy.

In an embodiment the present invention relates to a method of LATSinhibition in a cell or cell population using a compound of Formula A1or subformulae thereof or a salt thereof, or a stereoisomer thereof:

wherein X¹, X², ring A, R¹, R², R³, and R⁵ are as defined in thedetailed description infra.

Preferably the salt is a pharmaceutically acceptable salt. In a specificembodiment of the method of LATS inhibition in a cell populationaccording to the invention, the compound, or a salt thereof, is selectedfrom3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.In another specific embodiment of the method of LATS inhibition in acell population according to the invention, the compound, or a saltthereof, is selected fromN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

In another embodiment the present invention relates to a method of LATSinhibition in an ocular cell population using a compound of Formula A1or subformulae thereof or a salt thereof, or a stereoisomer thereof. Inyet another embodiment the present invention relates to a method of LATSinhibition in a cell population comprising limbal stem cells using acompound of Formula A1 or subformulae thereof or a salt thereof, or astereoisomer thereof. In yet a further embodiment the present inventionrelates to a method of LATS inhibition in a cell population comprisingcorneal endothelial cells using a compound of Formula A1 or subformulaethereof or a salt thereof, or a stereoisomer thereof.

Also preferably the method of LATS inhibition in a cell population isperformed ex vivo. In yet another preferred embodiment said compound ispresent in a concentration of 0.5 to 100 micromolar, preferably 0.5 to25 micromolar, more preferably 1 to 20 micromolar, particularlypreferably of about 3 to 10 micromolar. In one preferred embodiment ofthe method of LATS inhibition in a cell population comprising limbalstem cells the compound is present for 12 to 16 days, particularlypreferably the compound is present for 14 days. In another embodiment ofthe method of LATS inhibition in a cell population comprising cornealendothelial cells the compound is present for one to two weeks andsubsequently the cells are cultured for a period in growth mediumwithout supplementation with said compound, preferably wherein theperiod is one to two weeks. In an embodiment of the invention of themethod of LATS inhibition in a cell population the LATS inhibitorinhibits LATS1 or LATS2, or LATS1 and LATS2. In a more preferredembodiment the LATS inhibitor inhibits LATS1 and LATS2. In anotherpreferred embodiment of the method of LATS inhibition in a cellpopulation comprising limbal stem cells said method further comprisesgenetically modifying said limbal stem cells. In another preferredembodiment of the method of LATS inhibition in a cell populationcomprising corneal endothelial cells said method further comprisesgenetically modifying said corneal endothelial cells. Preferably saidgenetically modifying comprises introducing into said cell a geneediting system which specifically targets a gene associated withfacilitating a host versus graft immune response. In yet anotherpreferred embodiment the method of LATS inhibition in a cell population,the method comprises the further step after generation of an expandedpopulation of cells of rinsing those cells to substantially remove thecompound according to the invention. In one aspect the invention relatesto an expanded cell population comprising limbal stem cells obtainableby the method of LATS inhibition in a cell population comprising limbalstem cells according to the invention. In another aspect the inventionrelates to a an expanded cell population comprising limbal stem cellsobtained by the method of LATS inhibition in a cell populationcomprising limbal stem cells according to the invention. In one aspectthe invention relates to a population of corneal endothelial cellsobtainable by the method of LATS inhibition in a cell populationcomprising corneal endothelial cells according to the invention. Inanother aspect the invention relates to a population of cornealendothelial cells obtained by the method of LATS inhibition according tothe invention. In yet another aspect the invention relates to an ocularcell delivery preparation, comprising a cell population obtainable orobtained by the method of LATS inhibition according to the invention anda composition suitable for ocular delivery which is a localising agent.In a specific embodiment the localising agent is GelMa (which ismethacrylamide modified gelatin, and is also known as gelatinmethacrylate). In another specific embodiment the localising agent isfibrin or fibrin glue. Preferably the cell delivery preparation oflimbal stem cells has greater than 20% limbal stem cells. Alsopreferably the cell delivery preparation of limbal stem cells hasgreater than 20% p63alpha positive cells. In certain preferred aspectsthe cell population obtainable or obtained by the method of LATSinhibition according to the invention or cell delivery preparationaccording to the invention has only trace levels of the compoundaccording to the invention. Preferably, in the cell delivery preparationof corneal endothelial cells, corneal endothelial cells are present inthe cell delivery preparation at a density greater than 500 cells permm² (area). In certain preferred aspects the cell population obtainableor obtained by the method of LATS inhibition according to the inventionor cell delivery preparation according to the invention has only tracelevels of the compound according to the invention.

In another aspect the invention relates to a method of culturing cellscomprising culturing a population of cells in the presence of a LATSinhibitor. The cells can be a cell population as described and/or asprovided herein. Preferably the cells are ocular cells or liver cells.In a preferred embodiment the cells are ocular cells. In a furtheraspect the invention relates to a method of culturing cells comprisingculturing a population of cells comprising limbal stem cells in thepresence of a LATS inhibitor. In another aspect the invention relates toa method of culturing cells comprising culturing a population of cellscomprising corneal endothelial cells in the presence of a LATSinhibitor. In a preferred embodiment the invention relates to a methodof culturing cells comprising culturing a cell population comprisinglimbal stem cells, wherein the LATS inhibitor is a compound of FormulaA1 or subformulae thereof or salt thereof according to the invention. Inanother preferred embodiment the invention relates to a method ofculturing cells comprising culturing a population comprising cornealendothelial cells, wherein the LATS inhibitor is a compound of FormulaA1 or subformulae thereof or salt thereof according to the invention.Preferably the salt is a pharmaceutically acceptable salt. In apreferred embodiment said compound is present in a concentration of 0.5to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to20 micromolar, particularly preferably of about 3 to 10 micromolar. Inone preferred embodiment of the method of culturing cells comprisingculturing a cell population comprising limbal stem cells, the compoundis present for 12 to 16 days, particularly preferably the compound ispresent for 14 days. In another preferred embodiment of the method ofculturing cells comprising culturing a cell population comprisingcorneal endothelial cells, the compound is present for one to two weeksand subsequently the cells are cultured for a period in growth mediumwithout supplementation with said compound, preferably wherein theperiod is one to two weeks. In an embodiment of the invention the LATSinhibitor inhibits LATS1 or LATS2, or LATS1 and LATS2. In a more apreferred embodiment the LATS inhibitor inhibits LATS1 and LATS2. In oneembodiment said method further comprises genetically modifying cells.Preferably said genetically modifying comprises introducing into saidcell a gene editing system which specifically targets a gene associatedwith facilitating a host versus graft immune response. Preferably thecells are ocular cells. In one embodiment said method further comprisesgenetically modifying limbal stem cells. In another preferred embodimentsaid method further comprises genetically modifying corneal endothelialcells. In yet another preferred embodiment the method of culturing cellscomprises the further step after generation of an expanded population ofcells of rinsing those cells to substantially remove the compoundaccording to the invention. In one aspect the invention relates to anexpanded cell population obtainable by the method of culturing cellsaccording to the invention. In another aspect the invention relates toan expanded cell population obtained by the method of culturing cellsaccording to the invention. Preferably the cells are ocular cells. Inone aspect the invention relates to an expanded cell populationcomprising limbal stem cells obtainable by the method of culturing cellscomprising limbal stem cells according to the invention. In anotheraspect the invention relates to an expanded cell population comprisinglimbal stem cells obtained by the method of culturing cells comprisinglimbal stem cells according to the invention. In one aspect theinvention relates to a population of corneal endothelial cellsobtainable by the method of culturing cells comprising cornealendothelial cells according to the invention. In another aspect theinvention relates to a population of corneal endothelial cells obtainedby the method of culturing cells comprising corneal endothelial cellsaccording to the invention. In another aspect the invention relates toan ocular cell delivery preparation, comprising a cell populationobtainable or obtained by the method of culturing cells according to theinvention and a composition suitable for ocular delivery which is alocalising agent. In a specific embodiment the localising agent isGelMa. In another specific embodiment the localising agent is fibrin orfibrin glue. Preferably the cell delivery preparation of limbal stemcells has greater than 20% limbal stem cells. Also preferably the celldelivery preparation of limbal stem cells has greater than 20% p63alphapositive cells. Preferably corneal endothelial cells are present in thecell delivery preparation of corneal endothelial cells, at a densitygreater than 500 cells per mm² (area). In certain preferred aspects thecell population obtainable or obtained by the method of culturing cellsaccording to the invention or cell delivery preparation according to theinvention has only trace levels of the compound according to theinvention.

In another aspect the invention relates to a method of cell populationexpansion comprising the step of a) culturing a seeding population ofcells in the presence of a LATS inhibitor to generate an expandedpopulation of cells. In a preferred embodiment the method of cellpopulation expansion is performed ex vivo. Preferably the cells areocular cells or liver cells. In a preferred embodiment the cells areocular cells. In yet another aspect the invention relates to a method ofcell population expansion comprising the step of a) culturing a seedingpopulation of cells comprising limbal stem cells in the presence of aLATS inhibitor to generate an expanded population of cells comprisinglimbal stem cells. Preferably the LATS inhibitor is a compound ofFormula A1 or subformulae thereof or salt thereof, according to theinvention. In a further aspect the invention relates to a method of cellpopulation expansion comprising the step of a) culturing a seedingpopulation of cells comprising limbal stem cells in the presence of acompound of Formula A1 or subformulae thereof, or a salt thereof togenerate an expanded population of cells comprising limbal stem cells.In another embodiment of the invention said compound is selected fromFormula A2 or subformulae thereof or a salt thereof. In another aspectthe invention relates to a method of cell population expansioncomprising the step of a) culturing a seeding population of cellscomprising corneal endothelial cells in the presence of a LATS inhibitorto generate an expanded population of cells comprising cornealendothelial cells. Preferably the LATS inhibitor is a compound ofFormula A1 or subformulae thereof or salt thereof, according to theinvention. In a further aspect the invention relates to a method of cellpopulation expansion comprising the step of a) culturing a seedingpopulation of cells comprising corneal endothelial cells in the presenceof a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof to generate an expandedpopulation of cells comprising corneal endothelial cells. Preferablysaid compound is selected from Formula A2 or subformulae thereof. Alsopreferably the salt is a pharmaceutically acceptable salt. Preferablysaid compound is selected from the group of compounds consisting ofN-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propoxy)propan-2-ol;2,4-dimethyl-4-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)pentan-2-ol;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclobutyl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-isopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; and2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine.

Also preferably said compound or a salt thereof, is selected fromN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine and(S)—N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine.

In a preferred embodiment of the method of cell population expansionsaid compound is present in a concentration of 0.5 to 100 micromolar,preferably 0.5 to 25 micromolar, more preferably 1 to 20 micromolar,particularly preferably of about 3 to 10 micromolar. In one preferredembodiment of the method of cell population expansion relating to limbalstem cells in step a) the compound is present for 12 to 16 days,particularly preferably the compound is present for 14 days. In anotherpreferred embodiment of the method of cell population expansion relatingto corneal endothelial cells in step a) the compound is present for oneto two weeks and subsequently step b) is performed wherein the cells arecultured for a period in growth medium without supplementation with saidcompound, preferably wherein the period is one to two weeks. In aspecific embodiment of the method of cell population expansion relatingto limbal stem cells the compounds according to Formula A1 orsubformulae thereof produce greater than 30 fold expansion of the seededamount of cells. In another specific embodiment of the method of cellpopulation expansion relating to limbal stem cells the compoundsaccording to Formula A1 and subformulae thereof produce 100 fold to 2200fold, preferably 600 fold to 2200 fold expansion of the seeded amount ofcells. In an embodiment of the method of cell population expansionrelating to limbal stem cells, the method according to the inventionproduces a cell population with greater than 20% limbal stem cells. Inanother embodiment of the method of cell population expansion relatingto limbal stem cells, the method according to the invention produces acell population with greater than 50% limbal stem cells. In anotheraspect the method of cell population expansion relating to limbal stemcells according to the invention produces a cell population with greaterthan 20% expressing p63alpha. In yet another aspect the method of cellpopulation expansion relating to limbal stem cells according to theinvention produces a cell population with greater than 50% expressingp63alpha. In a specific embodiment of the method of cell populationexpansion relating to corneal endothelial cells the compounds accordingto Formula A1 or subformulae thereof produce greater than 10 foldexpansion of the seeded amount of cells. In another specific embodimentof the method of cell population expansion relating to cornealendothelial cells the compounds according to Formula A1 or subformulaethereof produce 15 fold to 600 fold, preferably 20 fold to 550 foldexpansion of the seeded amount of cells. In an embodiment of theinvention the LATS inhibitor inhibits LATS1 or LATS2, or LATS1 andLATS2. In a more preferred embodiment the LATS inhibitor inhibits LATS1and LATS2. In another preferred embodiment said method of cellpopulation expansion further comprises use of a gene editing system.Preferably said method comprises use of a gene editing system whichspecifically targets a gene associated with facilitating a host versusgraft immune response. Also preferably the cells are ocular cells orliver cells. In a preferred embodiment the cells are ocular cells. Inanother preferred embodiment said method of cell population expansionfurther comprises genetically modifying limbal stem cells, preferablywherein said genetically modifying comprises introducing into said cella gene editing system which specifically targets a gene associated withfacilitating a host versus graft immune response. In another preferredembodiment said method further comprises genetically modifying cornealendothelial cells, preferably wherein said genetically modifyingcomprises introducing into said cell a gene editing system whichspecifically targets a gene associated with facilitating a host versusgraft immune response. In yet another preferred embodiment, the methodof cell population expansion further comprises step c) rinsing theexpanded population of cells to substantially remove the compoundaccording to the invention.

In one aspect the invention relates to a kit comprising a LATSinhibitor, growth medium and instructions for cell population expansion.In another aspect the invention relates to a cell population obtainableby the method of cell population expansion according to the invention.In yet another aspect the invention relates to a cell populationobtained by the method of cell population expansion according to theinvention. In another aspect the invention relates to an ocular cellpopulation obtainable by the method of cell population expansionaccording to the invention. In another aspect the invention relates toan ocular cell population obtained by the method of cell populationexpansion according to the invention. In one aspect the inventionrelates to a cell population comprising limbal stem cells obtainable bythe method of cell population expansion relating to limbal stem cellsaccording to the invention. In another aspect the invention relates to acell population comprising limbal stem cells obtained by the method ofcell population expansion relating to limbal stem cells according to theinvention. In one aspect the invention relates to a cell populationcomprising corneal endothelial cells obtainable by the method of cellpopulation expansion relating to corneal endothelial cells according tothe invention. In another aspect the invention relates to a cellpopulation comprising corneal endothelial cells obtained by the methodof cell population expansion relating to corneal endothelial cellsaccording to the invention. In yet another aspect the invention relatesto an ocular cell delivery preparation, comprising a cell populationobtainable or obtained by the method of cell population expansionaccording to the invention and a composition suitable for oculardelivery which is a localising agent. In a specific embodiment thelocalising agent is GelMa. In another specific embodiment the localisingagent is fibrin or fibrin glue. Preferably the cell delivery preparationof limbal stem cells has greater than 20% limbal stem cells. Alsopreferably the cell delivery preparation of limbal stem cells hasgreater than 20% p63alpha positive cells. Preferably corneal endothelialcells are present in the cell delivery preparation of cornealendothelial cells, at a density greater than 500 cells per mm² (area).In certain preferred aspects the cell population obtainable or obtainedby the method of cell population expansion or cell delivery preparationaccording to the invention has only trace levels of the compoundaccording to the invention.

In some aspects of the method of cell population expansion, the methodfurther comprises use of a gene editing system. Preferably the geneediting system is used for genetically modifying cells. In embodimentsof methods according to the invention genetically modifying comprisesreducing or eliminating the expression and/or function of a geneassociated with facilitating a host versus graft immune response. Alsopreferably the gene editing system specifically targets a geneassociated with facilitating a host versus graft immune response.Preferably said gene editing system is selected from the groupconsisting of a CRISPR gene editing system, a TALEN gene editing system,a zinc finger nuclease gene editing system, a meganuclease gene editingsystem, AAV vector driven homologous recombination and lentiviralvectors-based genome editing technologies.

In one aspect the invention relates to an isolated cell population,wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofcells are limbal stem cells. Preferably greater than 20% are limbal stemcells. More preferably greater than 50% are limbal stem cells. Inanother preferred embodiment greater than 70% are limbal stem cells.Particularly preferably greater than 90% are limbal stem cells. In oneembodiment the cells have been gene edited.

In another aspect the invention relates to an isolated cell population,wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofcells are p63alpha expressing cells. Preferably greater than 20% arep63alpha positive. More preferably greater than 50% are p63alphapositive. In another preferred embodiment greater than 70% are p63alphapositive. Particularly preferably greater than 90% are p63alphapositive. In one embodiment the cells have been gene edited.

In a further aspect the invention relates to a cell populationcomprising limbal stem cells or the cell population according to theinvention, wherein one or more of said cells comprises a non-naturallyoccurring insertion or deletion of one or more nucleic acid residues ofa gene associated with facilitating a host vs graft immune response,wherein insertion and/or deletion results in reduced or eliminatedexpression or function of said gene. In a preferred embodiment said geneis selected from the group consisting of B2M, HLA-A, HLA-B and HLA-C. Ina specific embodiment the cells have genetically modified levels of B2Mexpression.

In a further aspect the invention relates to a cell populationcomprising corneal endothelial cells or the cell population according tothe invention, wherein one or more of said cells comprises anon-naturally occurring insertion or deletion of one or more nucleicacid residues of a gene associated with facilitating a host vs graftimmune response, wherein insertion and/or deletion results in reduced oreliminated expression or function of said gene. In a preferredembodiment said gene is selected from the group consisting of B2M,HLA-A, HLA-B and HLA-C. In a specific embodiment the cells havegenetically modified levels of B2M expression.

In a further aspect the invention relates to a cell populationcomprising limbal stem cells or corneal endothelial cells which havebeen gene edited. In a further aspect the invention relates to a cellpopulation comprising limbal stem cells which have been gene edited.Preferably the gene editing was performed by CRISPR. Preferably also theB2M gene was edited.

In one aspect the invention relates to a growth promoting agent of cellscomprising a LATS inhibitor. In one embodiment the invention relates toa growth promoting agent of ocular cells comprising a LATS inhibitor. Inone aspect the invention relates to a growth promoting agent of limbalstem cells comprising a LATS inhibitor. Preferably the LATS inhibitor isa compound of Formula A1 or subformulae thereof or salt thereof. Inanother aspect the invention relates to a growth promoting agent oflimbal stem cells comprising a compound of Formula A1 or subformulaethereof or salt thereof. In one aspect the invention relates to a growthpromoting agent of corneal endothelial cells comprising a LATSinhibitor. Preferably the LATS inhibitor is a compound of Formula A1 orsubformulae thereof or a salt thereof. In another aspect the inventionrelates to a growth promoting agent of corneal endothelial cellscomprising a compound of Formula A1 or subformulae thereof or a saltthereof.

In one aspect the invention relates to a pharmaceutical compositioncomprising a compound of Formula A2 or subformulae thereof, or apharmaceutically acceptable salt, or stereoisomer thereof, according tothe invention and at least one pharmaceutically acceptable excipient.Preferably the composition further comprises a preservation orcryopreservation solution.

In another aspect the invention relates to a cell proliferation mediumcomprising a LATS inhibitor and a growth medium. Preferably the LATSinhibitor is a compound of Formula A1 or subformulae thereof accordingto the invention. In one aspect the invention relates to a cellproliferation medium comprising a compound of Formula A1 or subformulaethereof according to the invention and a growth medium. In oneembodiment the cell proliferation medium additionally comprises cells asprovided herein. Preferably the cell proliferation medium additionallycomprises ocular cells. In another embodiment the cell proliferationmedium additionally comprises limbal stem cells. Preferably the limbalstem cells are in suspension. In yet another embodiment the cellproliferation medium comprises corneal endothelial cells. Preferably thecorneal endothelial cells are in suspension.

In one aspect the invention relates to a cell preparation comprising aLATS inhibitor and cells of a cell population as described and/orprovided herein. In another aspect the invention relates to a cellpreparation comprising a LATS inhibitor and ocular cells. In anotheraspect the invention relates to a cell preparation comprising a LATSinhibitor and limbal stem cells. In yet another aspect the inventionrelates to a cell preparation comprising a LATS inhibitor and cornealendothelial cells. Preferably the LATS inhibitor is a compound ofFormula A1 or subformulae thereof according to the invention. In anotheraspect the invention relates to a cell preparation comprising a compoundof Formula A1 according to the invention and limbal stem cells. In analternative aspect the invention relates to a cell preparationcomprising a compound of Formula A1 according to the invention andcorneal endothelial cells. Preferably the cell preparation furthercomprises a growth medium. Particularly preferably the cell preparationfurther comprises a preservation or cryopreservation solution.

In another aspect the invention relates to an ocular cell deliverypreparation, comprising a cell preparation according to the inventionand a composition suitable for ocular delivery which is a localisingagent. In a specific embodiment the localising agent is GelMa. Inanother specific embodiment the localising agent is fibrin or fibringlue. In another aspect the invention relates to an ocular cell deliverypreparation, comprising a cell preparation according to the inventionand a composition suitable for ocular delivery which is a localisingagent. In a specific embodiment the localising agent is GelMa. Inanother specific embodiment the localising agent is fibrin or fibringlue. In certain preferred aspects the cell preparation according to theinvention has only trace levels of the compound according to theinvention. In yet another specific embodiment greater than 20% of thecells in the cell delivery preparation are limbal stem cells. In afurther specific embodiment greater than 20% of the cells in the celldelivery preparation are p63alpha expressing cells.

In yet another aspect the invention relates to an ocular cell deliverypreparation, comprising a cell preparation according to the inventionand a composition suitable for ocular delivery which is a localisingagent. In a specific embodiment the localising agent is GelMa.

Preferably the corneal endothelial cells are in suspension. In analternative embodiment the corneal endothelial cells are present in thecell delivery preparation at a density greater than 500 cells per mm²(area). Particularly preferably the corneal endothelial cells arepresent at a density of 1000 to 3500 cells/mm² (area), more preferably2000 to about 3000 cells/mm² (area). In certain preferred aspects thecell preparation according to the invention has only trace levels of thecompound according to the invention.

Preferably the growth medium in the methods or cell preparationaccording to the invention is selected from the group consisting ofDulbecco's Modified Eagle's Medium (DMEM) supplemented with Fetal BovineSerum (FBS), human endothelial Serum Free (SF) Medium with human serum,X-VIVO15 medium and DMEM/F12 which is optionally supplemented withcalcium chloride; preferably X-VIVO15 medium.

Preferably the preservation or cryopreservation solution according tothe invention comprises a solution which is Optisol or PBS (phosphatebuffered saline) and the cryopreservation solution additionallycomprises glycerol, dimethyl sulfoxide, propylene glycol or acetamide.

In another aspect the invention relates to a kit comprising acomposition suitable for ocular delivery and a LATS inhibitor.Preferably the LATS inhibitor is a compound of Formula A2 or subformulaethereof according to the invention. In another aspect the inventionrelates to a kit comprising a composition suitable for ocular deliveryand compound of Formula A2 or subformulae thereof according to theinvention. Preferably the kit has instructions for use. In an embodimentthe composition suitable for ocular delivery is a localising agent ortopical eye drops. Preferably the composition suitable for oculardelivery is a localising agent. In a specific embodiment the kit furthercomprises limbal stem cells. In another specific embodiment of the kitthe composition suitable for ocular delivery is a localising agent whichis GelMa. In an alternative specific embodiment of the kit thecomposition suitable for ocular delivery is a localising agent which isfibrin or fibrin glue. In yet another specific embodiment, greater than20% of the cells in the kit are limbal stem cells. In a further specificembodiment, greater than 20% of the cells in the kit are p63alphaexpressing cells. In an alternative specific embodiment the kitcomprises corneal endothelial cells. In another specific embodiment thecomposition suitable for ocular delivery of corneal endothelial cells isa localising agent which is GelMa. In yet another specific embodimentcorneal endothelial cells are present in a monolayer. Preferably thecorneal endothelial cells are present at a density greater than 500cells per mm² (area). Particularly preferably the corneal endothelialcells are present at a density of 1000 to 3500 cells/mm² (area), morepreferably 2000 to about 3000 cells/mm² (area).

In preferred embodiments according to the invention the compositionsuitable for ocular delivery is a localising agent which is a biomatrix.Preferably the composition suitable for ocular delivery according to theinvention is a localising agent selected from the group consisting offibrin, collagen, gelatin, cellulose, amniotic membrane, fibrin glue,polyethylene (glycol) diacrylate (PEGDA), GelMA, localising agentscomprising a polymer, cross-linked polymer, or hydrogel comprising oneor more of hyaluronic acid, polyethylene glycol, polypropylene glycol,polyethylene oxide, polypropylene oxide, poloxamer, polyvinyl alcohol,polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone,poly(lactide-co-glycolide), alginate, gelatin, collagen, fibrinogen,cellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl-guar, gellan gum,guar gum, xanthan gum and carboxymethylcellulose, as well as derivativesthereof, co-polymers thereof, and combinations thereof. In preferredembodiments according to the invention the composition suitable forocular delivery is a localising agent which is GelMa, fibrin or fibringlue. In specific embodiments according to the invention the compositionsuitable for ocular delivery is a localising agent which is GelMa. Inother specific embodiments according to the invention the compositionsuitable for ocular delivery is a localising agent which is fibrin orfibrin glue. Preferably the localising agent is fibrin glue. Fibringlues are known in the art, including, for example, TISSEEL VH Fibrinsealant (Baxter A G, Vienna, Austria) (Panda et al., 2009, Indian JOphthalmol. September-October; 57(5): 371-379). In one embodiment fibringlue is used for the delivery of limbal stem cells. In anotherembodiment GelMa is used for the delivery of corneal endothelial cells.

In more preferred embodiments according to the invention wherein limbalstem cells are present in combination with the localising agent, greaterthan 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are limbalstem cells. Preferably greater than 20% are limbal stem cells. Morepreferably greater than 50% are limbal stem cells. In another preferredembodiment greater than 70% are limbal stem cells. Particularlypreferably greater than 90% are limbal stem cells.

In further preferred embodiments according to the invention whereincells are present in combination with the localising agent, greater than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are p63alphaexpressing cells. Preferably greater than 20% are p63alpha positivecells. More preferably greater than 50% are p63alpha positive cells. Inanother preferred embodiment greater than 70% are p63alpha positivecells. Particularly preferably greater than 90% are p63alpha positivecells.

In more preferred embodiments according to the invention cornealendothelial cells are present in combination with the localising agent.Preferably the corneal endothelial cells are in a monolayer. Morepreferably the corneal endothelial cells are present at a densitygreater than 500 cells per mm² (area). Particularly preferably thecorneal endothelial cells are present at a density of 1000 to 3500cells/mm² (area), more particularly preferably 2000 to about 3000cells/mm² (area).

In further particularly preferred embodiments of the invention the LATSinhibitor inhibits LATS1 or LATS2, or LATS1 and LATS2. In a moreparticularly preferred embodiments according to the invention the LATSinhibitor inhibits LATS1 and LATS2.

In preferred embodiments according to the invention the compound isselected from the group consisting of2-methyl-1-(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propoxy)propan-2-ol;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;(S)—N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)pentan-2-ol;N-isopropyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclobutyl)pyrido[3,4-d]pyrimidin-4-amine;N-isopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-1-ol;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propoxy)ethan-1-ol;and(R)—N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine.

Also preferably said compound or a salt thereof, is selected fromN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine and(S)—N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine.Preferably said compound or a salt thereof, isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

In particularly preferred embodiments according to the invention thecompound according to the invention is present in a concentration of 0.5to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to20 micromolar, particularly preferably of about 3 to 10 micromolar.

The invention relates in one aspect to a method of transplanting apopulation of cells to a subject, said method comprising administeringthe population of cells obtainable or obtained by the method of cellpopulation expansion or method of culturing cells or method of LATSinhibition according to the invention.

The invention further relates to a method of transplanting a populationof ocular cells onto the eye of a subject, said method comprisingadministering the population of cells obtainable or obtained by themethod of cell population expansion or method of culturing cells ormethod of LATS inhibition according to the invention, wherein the cellsare ocular cells. Preferably ocular cells are limbal stem cells orcorneal endothelial cells. The invention relates in another aspect to amethod of transplanting a population of ocular cells onto the cornea ofa subject, said method comprising administering the cell deliverypreparation according to the invention.

The invention relates in another aspect to a method of transplanting apopulation of cells comprising limbal stem cells onto the cornea of asubject, said method comprising administering the population of cellscomprising limbal stem cells obtainable or obtained by the method ofcell population expansion or method of culturing cells or method of LATSinhibition according to the invention. The invention relates in anotheraspect to a method of transplanting a population of cells comprisinglimbal stem cells onto the cornea of a subject, said method comprisingadministering the cell delivery preparation according to the invention.

The invention relates in another aspect to a method of transplanting acell population comprising limbal stem cells onto the cornea of asubject, said method comprising expanding a cell population comprisinglimbal stem cells by culturing said population with cell proliferationmedium comprising a LATS inhibitor according to the invention,preferably rinsing the expanded cell population to substantially removethe LATS inhibitor, and administering said cells onto the cornea of saidsubject. Preferably said cell population is combined with a biomatrixprior to said administration. In a specific embodiment said cellpopulation is combined with a biomatrix which is GelMA prior to saidadministration. In a another specific embodiment said cell population iscombined with fibrin glue prior to said administration. In an embodimentsaid cell population is combined with a carrier which is a contact lens.In a specific embodiment the cell population comprising limbal stemcells is combined with a biomatrix which is GelMA and the GelMA ispolymerized on a carrier which is a contact lens. In another specificembodiment the cell population comprising limbal stem cells is combinedwith fibrin glue and a contact lens.

The invention relates in one aspect to a method of transplanting apopulation of corneal endothelial cells onto the cornea of a subject,said method comprising administering the population of cornealendothelial cells obtainable or obtained by the method of cellpopulation expansion or method of culturing cells or method of LATSinhibition according to the invention. The invention relates in anotheraspect to a method of transplanting a population of corneal endothelialcells onto the cornea of a subject, said method comprising administeringthe cell delivery preparation according to the invention.

The invention relates in another aspect to a method of transplanting apopulation of cells comprising corneal endothelial cells onto the corneaof a subject, said method comprising expanding a population of cellscomprising corneal endothelial cells by culturing said population withcell proliferation medium comprising a LATS inhibitor according to theinvention, rinsing the expanded population of cells to substantiallyremove the LATS inhibitor, and administering said cells onto the corneaof said subject. Preferably said cells are combined with a biomatrixprior to said administration. In a specific embodiment said cells arecombined with a biomatrix which is GelMA prior to said administration.In a more specific embodiment said corneal endothelial cells arecombined with a biomatrix which is bioprinted onto the ocular surface.Particularly preferably said corneal endothelial cells are combined witha biomatrix which is GelMA and bioprinted onto the ocular surface bypolymerising the GelMA by a light triggered reaction.

The invention relates in another aspect to a method of transplanting apopulation of cells to the eye of a subject, comprising combining thecells with a biomatrix to form a cell/biomatrix mixture, injecting themixture into the eye of the subject or applying the mixture onto thesurface of the eye of the subject, and bioprinting the cells in or onthe eye by guiding and fixing the cells, such as on the cornea, using alight source, such as an Ultraviolet A or white light source. In certainembodiments, the light source produces light of a wavelength that is atleast 350 nm. In certain embodiments, the light source produces light inthe 350 nm to 420 nm range. For example, an LED light source can be usedto produce a light having a wavelength of 365 nm or 405 nm, or any otherwavelength above 350 nm, or a mercury lamp with a bandpass filter can beused to produce a light having a wavelength of 350 nm to 700 nm, forexample a wavelength of 365 nm or 405 nm. In another embodiment, thelight source produces visible, white light having a wavelength, forexample, in the 400 nm to 700 nm range. In certain embodiments, thecells are ocular cells, such as corneal cells, for example cornealendothelial cells.

The invention relates in another aspect to a method of transplanting apopulation of corneal endothelial cells to the eye of a subject,comprising culturing a population of corneal endothelial cells in a cellproliferation medium that comprises a LATS inhibitor, combining thecorneal endothelial cells with a biomatrix to form a cell/biomatrixmixture, injecting the mixture into the eye of the subject, andbioprinting the cells in the eye by guiding and fixing the cells on thecornea using a light source, such a UVA or LED or visible light source.

The invention relates in a further aspect to a method of prophylaxis ortreatment of an ocular disease or disorder using a LATS inhibitor.Preferably the LATS inhibitor is a compound of Formula A1 or subformulaethereof according to the invention. The invention relates in yet afurther aspect to a method of prophylaxis or treatment of an oculardisease or disorder using a compound according to Formula A2 orsubformulae thereof according to the invention. In preferred specificembodiments the method of prophylaxis or treatment of an ocular diseaseor disorder further comprises the method of LATS inhibition in a cellpopulation or the method of cell population expansion according to theinvention, wherein said cells are ocular cells. Preferably the method ofprophylaxis or treatment of an ocular disease or disorder comprisesadministering to a subject in need thereof of a therapeuticallyeffective amount of a cell population obtainable or obtained by themethod of cell population expansion according to the invention, whereinsaid cells are ocular cells. In another preferred embodiment the methodof prophylaxis or treatment of an ocular disease or disorder comprisesadministering to a subject in need thereof of a therapeuticallyeffective amount of the cell delivery preparation according to theinvention, wherein said cells are ocular cells. In yet another preferredembodiment the method of prophylaxis or treatment of an ocular diseaseor disorder the method comprises the steps of the method oftransplanting a population of cells comprising ocular cells to the eyeof a subject according to the invention. Preferably the ocular cells arelimbal stem cells or corneal endothelial cells. In yet another preferredembodiment the method of prophylaxis or treatment of an ocular diseaseor disorder the method comprises the steps of the method oftransplanting a population of cells comprising limbal stem cells ontothe cornea of a subject according to the invention. In an alternativelypreferred embodiment the method of prophylaxis or treatment of an oculardisease or disorder the method comprises the steps of the method oftransplanting a population of corneal endothelial cells onto the corneaof a subject according to the invention. In a specific embodiment of themethod of prophylaxis or treatment of an ocular disease or disorderaccording to the invention the cell population obtainable or obtained bythe method of cell population expansion according to the invention orcell delivery preparation according to the invention is administeredsimultaneously or sequentially with an agent or agents selected from thegroup consisting of dexamethasone, cyclosporine, tobramycin, andcefazolin.

In one aspect the invention relates to a YAP (yes associated protein)modulator for use in a method of transplanting a population of cells toa subject, which comprises administering to a subject in need thereof ofa therapeutically effective amount of a cell population obtainable orobtained by the method of cell population expansion according to theinvention or cell delivery preparation according to the invention.Preferably said YAP modulator is a LATS inhibitor. Preferably the cellsare ocular cells.

In one aspect the invention relates to a YAP (yes associated protein)modulator for use in a method of transplanting a population of cellscomprising limbal stem cells onto the cornea of a subject, whichcomprises administering to a subject in need thereof of atherapeutically effective amount of a cell population obtainable orobtained by the method of cell population expansion according to theinvention or cell delivery preparation according to the invention.Preferably said YAP modulator is a LATS inhibitor.

In another aspect the invention relates to a YAP modulator for use in amethod of treating limbal stem cell deficiency, comprising administeringto a subject in need thereof of a therapeutically effective amount of acell population obtainable or obtained by the method of cell populationexpansion according to the invention or cell delivery preparationaccording to the invention. Preferably said YAP modulator is a LATSinhibitor.

In one aspect the invention relates to a YAP (yes associated protein)modulator for use in a method of transplanting a population of cornealendothelial cells onto the cornea of a subject, which comprisesadministering to a subject in need thereof of a therapeuticallyeffective amount of a cell population obtainable or obtained by themethod of cell population expansion according to the invention or celldelivery preparation according to the invention. Preferably said YAPmodulator is a LATS inhibitor.

In another aspect the invention relates to a YAP modulator for use in amethod of treating corneal endothelial dysfunction, comprisingadministering to a subject in need thereof of a therapeuticallyeffective amount of a cell population obtainable or obtained by themethod of cell population expansion according to the invention or celldelivery preparation according to the invention. Preferably said YAPmodulator is a LATS inhibitor.

In yet another embodiment, the present invention relates to a method oftreatment of a disease or disorder comprising administering to a subjectin need thereof a cell population, wherein the population has been grownin the presence of an agent capable of inhibiting the activity of LATS1and LATS2 kinases; thereby inducing YAP translocation and drivingdownstream gene expression for cell proliferation. In a furtherembodiment, the agent is a compound of Formula A1 or subformulaethereof, or a pharmaceutically acceptable salt thereof. Preferably thecells are ocular cells.

In one aspect the invention relates to a compound according to FormulaA2 or subformulae thereof, or a pharmaceutically acceptable salt thereofaccording to the invention for use in therapy or as a medicament.Preferably the compound is for use in an ocular disease or disorder.

In another aspect the invention relates to a LATS inhibitor for use inan ocular disease or disorder, preferably wherein the LATS inhibitor isa compound. Preferably the compound is a compound of Formula A1 orsubformulae thereof, or a pharmaceutically acceptable salt thereofaccording to the invention.

In yet another aspect the invention relates to the use of a compound ofFormula A2 or subformulae thereof, or a pharmaceutically acceptable saltthereof according to the invention in the manufacture of a medicament.In a further aspect the invention relates to the use of a compound ofFormula A1 or subformulae thereof, or a pharmaceutically acceptable saltthereof according to the invention in the manufacture of a medicament totreat an ocular disease or disorder.

In preferred specific embodiments of the compound for use according tothe invention or LATS inhibitor for use according to the invention oruse of the compound in a manufacture of a medicament according to theinvention, the use further comprises the method of LATS inhibition in acell population or the method of cell population expansion according tothe invention.

In further preferred specific embodiments of the compound for useaccording to the invention or LATS inhibitor for use according to theinvention or use of the compound in a manufacture of a medicamentaccording to the invention, the use comprises administering to a subjectin need thereof a therapeutically effective amount of a cell populationobtainable or obtained by the method of cell population expansionaccording to the invention. In yet further preferred embodiments of thecompound for use according to the invention or LATS inhibitor for useaccording to the invention or use of the compound in a manufacture of amedicament according to the invention, the use comprises administeringto a subject in need thereof of a therapeutically effective amount ofthe cell delivery preparation according to the invention. In onepreferred embodiment of the compound for use according to the inventionor LATS inhibitor for use according to the invention or use of thecompound in a manufacture of a medicament according to the invention,the use comprises the steps of the method of transplanting a populationof cells comprising ocular cells onto the cornea of a subject accordingto the invention. In yet more preferred embodiments of the compound foruse according to the invention or LATS inhibitor for use according tothe invention or use of the compound in a manufacture of a medicamentaccording to the invention, the use comprises the steps of the method oftransplanting a population of cells comprising limbal stem cells ontothe cornea of a subject according to the invention. In yet morepreferred embodiments of the compound for use according to the inventionor LATS inhibitor for use according to the invention or use of thecompound in a manufacture of a medicament according to the invention,the use comprises the steps of the method of transplanting a populationof cells comprising corneal endothelial cells onto the cornea of asubject according to the invention. In specific embodiments of thecompound for use according to the invention or LATS inhibitor for useaccording to the invention or use of the compound in a manufacture of amedicament according to the invention, the cell population obtainable orobtained by the method of cell population expansion according to theinvention or cell delivery preparation according to the invention isadministered simultaneously or sequentially with an agent or agentsselected from the group consisting of dexamethasone, cyclosporine,tobramycin, and cefazolin.

In preferred embodiments according to the invention, the ocular diseaseor disorder is associated with limbal stem cell deficiency. In morepreferred embodiments the ocular disease or disorder is limbal stem celldeficiency. More preferably the ocular disease or disorder is limbalstem cell deficiency which arises due to an injury or disorder selectedfrom the group consisting of chemical burns, thermal burns, radiationinjury, aniridia, sclerocornea, multiple endocrine neoplasia, StevensJohnson syndrome, ocular cicatricial pemphigoid, collagen vasculardiseases; chronic non-auto-immune inflammatory disorders arising fromcontact lens use, dry eye disease, rosacea, staph marginal, keratitis(including bacterial, fungal & viral keratitis), pterygia or neoplasm,limbal stem cell deficiency arising after multiple eye surgeries,excision of pterygia or neoplasm or cryotherapy; and limbal stem celldeficiency arising as a result of medication toxicity from a medicationselected from the group consisting of preservatives (thimerosal,benzalkonium), topical anesthetics, pilocarpine, beta blockers,mitomycin, 5-fluorouracil, silver nitrate, and oral medications causingStevens Johnson syndrome. Particularly preferably the ocular disease ordisorder is limbal stem cell deficiency which arises due to an injury ordisorder selected from the group consisting of chemical burns, aniridia,Stevens Johnson Syndrome and contact lens use.

In preferred embodiments according to the invention, the ocular diseaseor disorder is associated with decreased corneal endothelial celldensity. In more preferred embodiments the ocular disease or disorder iscorneal endothelial dysfunction. More preferably the ocular disease ordisorder is corneal endothelial dysfunction which is selected from thegroup consisting of Fuchs endothelial corneal dystrophy, bullouskeratopathy (including pseudophakic bullous keratopathy and aphakicbullous keratopathy), corneal transplant failure, posterior polymorphouscorneal dystrophy, congenital hereditary endothelial dystrophy, X-linkedendothelial corneal dystrophy, aniridia, and corneal endothelitis. In aspecific embodiment the ocular disease or disorder is selected from thegroup consisting of Fuchs endothelial corneal dystrophy, bullouskeratopathy (including pseudophakic bullous keratopathy and aphakicbullous keratopathy) and corneal transplant failure.

The invention further relates to methods of promoting wound healing,particularly for treating or ameliorating the symptoms of burns, acuteand chronic skin ulcers, comprising administering to a subject in needthereof an effective amount of a LATS inhibitor.

Within certain other aspects, the invention relates to a method ofpromoting wound healing comprising administering a therapeuticallyeffective amount of a compound of Formula A1 or subformulae thereof, ora pharmaceutically acceptable salt, or a stereoisomer thereof.

In another aspect, the invention relates to compounds and compositionsthat may be used in promoting wound healing. In another aspect, theinvention relates to compounds and compositions that may be used for themanufacture of a medicament for promoting wound healing.

The present invention also relates to a method of promoting woundhealing, particularly for treating or ameliorating the symptoms ofburns, acute skin ulcers, and chronic skin ulcers, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula A1 or subformulae thereof, andoptionally with a second therapeutic agent that is another compound ofthe invention or one other type of therapeutic agent.

The present invention also relates to a method of promoting ocular woundhealing comprising administering to an eye of a subject atherapeutically effective amount of a compound of the invention. In oneembodiment, the ocular wound is a corneal wound. In other embodiments,the ocular wound is an injury or surgical wound.

In another aspect, the invention relates to compounds and compositionsthat may be used in liver regeneration and liver regrowth. Withincertain other aspects, the invention relates to a method of promotingliver regeneration and liver regrowth comprising administering atherapeutically effective amount of a compound of Formula A1 orsubformulae thereof, or a pharmaceutically acceptable salt thereof, or astereoisomer thereof. In another aspect, the invention relates tocompounds and compositions that may be used for the manufacture of amedicament for liver regeneration and liver regrowth.

The present invention also relates to a method for liver regenerationand liver regrowth, particularly for treatment of insufficient liverregrowth following transplantation of marginal grafts; for supportingenhanced regrowth of the remnant liver mass following extensivehepatectomy; for regeneration of patients' of livers following acuteliver failure from viral hepatitis, drug-induced liver injury,autoimmune hepatitis, ischemic- and congestive liver disease; and fortreatment of patients with chronic liver injury and underlying liverfibrosis, from non-alcoholic steatohepatitis, alcoholic steatohepatitis,chronic viral hepatitis B and C, hemochromatosis, alpha-1 anti-trypsindeficiency, Wilson's disease and drug-induced liver fibrosis to enhanceboth regenerative capacity and accelerate fibrosis resolution,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound of the present invention and optionallywith a second therapeutic agent that is another compound of theinvention or one other type of therapeutic agent.

In certain embodiments, the invention relates to a method of generatingcellular material for cell therapy and/or transplantation comprising theex-vivo use of a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof, or a stereoisomer thereof. Thecellular material may comprise ocular, liver or skin cells.

Furthermore, in certain embodiments, the invention relates to a methodof promoting liver regeneration and liver regrowth comprising theex-vivo use of a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof, or a stereoisomer thereof.

The present invention also relates to an ex-vivo method for liver cellpopulation expansion, comprising use of a compound of the presentinvention or a pharmaceutically acceptable salt thereof, or astereoisomer thereof.

Other features and advantages of the present invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : LATS inhibitors (compound ex. 49 and ex. 133) induce YAPdephosphorylation in LSCs within one hour of treatment as shown byWestern blot.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D: Immunolabelling of p63-alpha inlimbal stem cell cultures indicates that the LSC population can beexpanded when it is maintained in medium comprising the LATS inhibitors(compound ex. 49 and ex. 133). FIG. 2A: In the presence of growth mediumand DMSO, only a few isolated cells attach to the culture dish andsurvive up to 6 days. Most cells expressed the human nuclear marker, butfew expressed p63alpha. FIG. 2B and FIG. 2C: In contrast, in thepresence of LATS inhibitors: compound example no. 49 and example no.133, the cells formed colonies and expressed p63alpha. This resultindicated that the LATS inhibitors promote the expansion of thepopulation of cells with the p63alpha-positive phenotype. FIG. 2D:Passaging cells and culturing them in the presence of LATS inhibitorcompound example no. 49 for two weeks enabled cell population expansionand the formation of confluent cultures expressing p63alpha.

FIG. 3 : LATS1 and LATS2 knockdown by siRNA activates LSC proliferationin culture as shown by percentage of EdU positive cells.

FIG. 4 : Immunolabelling of LSC markers DeltaN-p63 alpha/beta/gamma,ABCG2 and C/EBP delta indicates that LSCs maintained in a culture mediumcontaining the LATS inhibitors (compound ex. 49 and ex. 133) expresstypical markers of LSCs. Results indicate that cells cultured in DMSO donot typically express markers such as DeltaN-p63 alpha/beta/gamma, ABCG2and C/EBP delta, normally expressed by LSCs. In contrast, cells culturedin the presence of LATS inhibitors express markers such as DeltaN-p63alpha/beta/gamma, ABCG2 and C/EBP delta, normally expressed by LSCs.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D: Immunolabelling of undifferentiatedLSC marker p63alpha and corneal epithelium cell marker keratin 12 showsthat LSC populations expanded using a culture medium comprising the LATSinhibitors (FIG. 5A: compound ex. 49, FIG. 5B: compound ex. 47, FIG. 5C:compound ex. 12, FIG. 5D: compound ex. 261) can differentiate intocorneal epithelium cells when transferred to conditions that enabledifferentiation. Shown are views where the transition from thep63alpha-positive cell identity to the keratin-12 identity areoccurring.

FIG. 6 : LSCs were labeled using a fluorescent protein in order toconfirm that LSCs attached to a contact lens using GelMA polymerizationcan be delivered to the surface of the rabbit eye ex vivo. Arrows showsite of attachment of LSCs.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D: FIG. 7A: Transplanted eye,keratin-12 staining; FIG. 7B: Transplanted eye, keratin-19 staining;FIG. 7C: non-transplanted control eye, keratin-12 staining; FIG. 7D:non-transplanted control eye, keratin-19 staining. These figures showthat in a rabbit model of limbal stem cell deficiency, a population ofLSCs expanded in medium comprising compound example no. 12, combinedwith GelMA and delivered via a contact lens in vivo to the rabbit'scorneal surface, lead to regeneration of a keratin-12-positive cornealepithelium (FIG. 7A) and prevented conjunctivalization bykeratin-19-positive conjunctival cells in the transplanted eye. FIG. 7B:Arrow is pointing to absence of keratin 19 staining. In contrast,non-transplanted rabbit eyes showed absence of keratin-12-positivecorneal epithelium restoration. FIG. 7C: Arrow is pointing to absence ofkeratin-12 staining. Instead, signs of conjunctivalization wereobserved, as showed by the presence of keratin-19 staining. FIG. 7D:Arrow is pointing to areas of positive keratin-19 staining.

FIG. 8A, FIG. 8B: FIG. 8A: Rabbit eye transplanted with human LSCs; FIG.8B: Control rabbit eye, non-transplanted with human LSCs. These Figuresshow that the cells that restored the corneal epithelium of transplantedeyes were human (FIG. 8A), as demonstrated by the presence of humanmitochondrial protein (arrow shows the human mitochondrial marker ispresent). In contrast, the ocular surface of non-transplanted eyes didnot exhibit human mitochondrial protein staining (FIG. 8B: humanmitochondrial marker is absent).

FIG. 9 : Microscopic images of LSCs delivered via TISSEEL to collagencoated 24 well plate show repopulation of the cells to cover the culturesurface within 2 weeks.

FIG. 10 : CellTracker Green CMFDA labeled LSCs were delivered to humancornea ex vivo and covered with protective contact lens.

FIG. 11 : Red and green dye labeled HEK-293 cells were bioprinted into aYin-Yang pattern on top of a rabbit cornea ex vivo (shown as dark andlight gray pattern).

FIG. 12 : Reducing immune rejection by CRISPR/Cas9-mediated deletion ofthe beta-2-microglobulin (B2M) gene in LSCs: FACS analyses show thatCRISPR-mediated deletion of B2M and subsequent elimination of HLA A, Band C occurred in 21 percent of the LSCs.

FIG. 13 : The population of B2M-negative/HLA A,B,C-negative LSCs wasexpanded using compound example no. 48a to produce a cell preparationwhere 97 percent of the cells do not express HLA A, B, C.

FIG. 14 : LATS inhibitors (compounds ex. 133 and ex. 49) inducetranslocation of YAP into the nucleus in Corneal Endothelial Cells(CECs).

FIG. 15A, FIG. 15B, FIG. 15C: LATS inhibitors (compound ex. 133 and ex.49) induce YAP dephosphorylation in CECs within one hour of treatment.As shown in FIG. 15A by Western blot; FIG. 15B: graph showingphosphorylated YAP levels normalized to beta-actin; and FIG. 15C: graphshowing phosphorylated YAP levels normalized to total YAP.

FIG. 16 : CECs grown in presence or absence of LATS inhibitors. AnIncucyte system (Essen Biosciences) was used to measure CEC confluenceby real-time quantitative live-cell analysis over a time course.Compound ex. 49 (black squares) and ex. 133 (light grey squares) inducedstrong CEC proliferation; whereas CEC proliferation was minimal in thevehicle (DMSO, dark grey squares).

FIG. 17 : LATS1 and LATS2 knockdown by siRNA activates cornealendothelial cell proliferation in culture as shown by percentage of EdUpositive cells.

FIG. 18A, FIG. 18B: Zonula Occludens-1 (ZO-1) immunolabelling indicatesthat CECs proliferated in the presence of the LATS inhibitor, compoundex. 49, (FIG. 18A) form tight junctions, an endothelial structure andretain a normal cell size and morphology characteristic of functionalCECs. CECs proliferated in the presence of the vehicle alone (DMSO) showsigns of polymegatism characteristic of dysfunctional CECs (FIG. 18B).

FIG. 19 : Quantitative R T-PCR analysis indicates that a cornealendothelial cell population expanded with the LATS inhibitor, compoundex. 49, express genes normally expressed by corneal endothelial cells invivo, including Collagen 8a2, AQP1, SLc4A11. The cells do not expressmarkers of other epithelia present in the eye, including RPE65 (a markerof retinal pigmented epithelium) and CD31 (a marker of vascularepithelium).

FIG. 20A, FIG. 20B: Immunohistochemical analysis indicates that acorneal endothelial cell population expanded with the LATS inhibitor,compound ex. 49, express genes normally expressed by corneal endothelialcells in vivo, including Na/K ATPase (FIG. 20A) and Collagen 8a2 (FIG.20B).

FIG. 21A, FIG. 21B, FIG. 21C, FIG. 21D: FACS analysis of the cornealendothlelium cell population expanded in the presence of the LATSinhibitor, compound ex. 47, and CECs cultured in the absence of a LATSinhibitor. The cell population expanded in the presence of the LATSinhibitor expresses low levels of CD73 (FIG. 21A, grey line), CD44 (FIG.21B, grey line), CD166 (FIG. 21C, grey line) and CD105 (FIG. 21D, greyline); while the cell population cultured without the LATS inhibitorexpresses high levels of CD44, CD73, CD105 and CD166 (black lines).

FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D: Bubble depression methoddepicting method 1 as described further under the “bio-printingsection”, in which a biomatrix is applied to an eye to deliver a cellpreparation according to the invention. FIG. 22A. Inject bolus ofbiomatrix; FIG. 22B Inject bubble beneath biomatrix to spread overcornea; FIG. 22C Cure biomatrix with UV or blue light source; FIG. 22DRemove bubble and replace with balanced salt solution.

FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, FIG. 23E: Subtractive methodusing femtosecond (FS) laser, depicting method 2 as described furtherunder the “bio-printing section”, in which a biomatrix is applied to aneye to deliver a cell preparation according to the invention. FIG. 23ARemove dysfunctional endothelium with FS; FIG. 23B Inject biomatrix inanterior chamber; FIG. 23C Cure biomatrix with UV or blue light source;FIG. 23D Detach unwanted biomatrix with FS; FIG. 23E Remove detachedbiomatrix with forceps.

FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D, FIG. 24E: Dye mask method,depicting method 3 as described further under the “bio-printingsection”, in which a biomatrix is applied to an eye to deliver a cellpreparation according to the invention. FIG. 24A Stain the endotheliumwith dye (e.g. Typan Blue); FIG. 24B Peel the dysfunctional endothelium;FIG. 24C Inject dyed biomatrix FIG. 24D Cure biomatrix with UV or bluelight source; FIG. 24E Flush uncured biomatrix.

FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D: Dry dispense method, depictingmethod 4 as described further under the “bio-printing section”, in whicha biomatrix is applied to an eye to deliver a cell preparation accordingto the invention. FIG. 25A Drain anterior chamber; FIG. 25B Dispensebiomatrix to posterior cornea with soft tip/brush cannula; FIG. 25C Curebiomatrix with UV or blue light source; FIG. 25D Refill eye withbalanced salt solution.

FIG. 26 Schematic showing bioprinting device as described further inExample C11.

FIG. 27 Results showing cells can be bioprinted on the posterior side ofthe cornea by using a handheld device that projects 365 nm UVA lightthrough the cornea. After unpolymerized and unattached material wasrinsed, a circular pattern of fluorescent protein-labelled cells wasretained on the posterior side of the cornea.

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, FIG. 28E, FIG. 28F: CECsbioprinted on the posterior side of the cornea can rebuild a cornealendothelium in a rabbit model of corneal endothelium dystrophy. Resultsindicated that in experimental rabbits, the corneal endotheliumstructure can be detected using ZO-1 immunohistochemistry (FIG. 28A). Inthe right eye of a rabbit where the corneal endothelium was surgicallyremoved and no CEC was bioprinted, the ZO-1 staining is absent,indicating an absence of normal corneal endothelium structure (FIG.28B). In the right eye of a rabbit where the corneal endothelium wassurgically removed and CEC were bioprinted, the ZO-1 staining ispresent, indicating that a corneal endothelium structure has beenrebuilt (FIG. 28C). FIG. 28D and FIG. 28E show that human nuclearantigen immunostaining is absent in eyes that did not receive any humanCECs. In contrast, human nuclear antigen-positive cells cover the imagedfield in eyes where human CECs were bioprinted (FIG. 28F), indicatingthat the ZO-1-labeled corneal endothelium structure shown in FIG. 28C iscomposed of the human CECs bioprinted on the posterior side of therabbit cornea.

FIG. 29 : Red-fluorescent-protein labeled HEK-293 cells were bioprintedinto constructs of different letters on the posterior side of humancornea ex vivo.

FIG. 30 : Vector map shows the design of the AAV2 vector used to expressthe CRISPR system in LSCs and CECs. (FIG. 30 discloses SEQ ID NO: 28).

FIG. 31 : FACS analysis of AAV-mediated expression of the CRISPR systemenabled deletion of B2M and subsequent elimination of HLA A, B and C inLSCs.

FIG. 32 : FACS analysis of AAV-mediated expression of the CRISPR systemenabled deletion of B2M and subsequent elimination of HLA A, B and C inCECs.

FIG. 33A: FIG. 33A is a western blot of pYAP in lysate of human HaCaTcells that were untreated or treated by 40 pM each of siRNA againstMST1/2 or LATS1/2; actin was used as control.

FIG. 33B: FIG. 33B is a western blot of pYAP in lysate of human HaCaTcells that were untreated or treated by 9 μM of Example 133; actin wasused as control.

FIG. 33C: FIG. 33C is a graph of the relative inhibitory activityagainst LATS1 versus concentration of Example 133 ranging from ˜10⁻⁴ to1 μm. The calculated IC₅₀ of Example 133 against LATS1 was 1.3 nM.

FIG. 34 : is a bar graph of relative Cyr61/Gapdh expression levelsversus concentration of Example 133 at 0, 0.2 and 2 mg/mL.

FIG. 35A: FIG. 35A shows micrographs of mouse skin treated topicallywith vehicle or Example 133.

FIG. 35B: FIG. 35B is a scatter plot comparing the percentage of Ki67+cells in mouse skin treated with vehicle or Example 133.

DETAILED DESCRIPTION OF THE INVENTION

LATS

LATS is the abbreviated name of the large tumor suppressor kinase. LATSas used herein refers to LATS1 and/or LATS2. LATS1 as used herein refersto the large tumor suppressor kinase 1 and LATS2 refers to the largetumor suppressor kinase 2. LATS1 and LATS2 both have serine/threonineprotein kinase activity. LATS1 and LATS2 have been given the HumanGenome Organisation (HUGO) Gene Nomenclature Committee identifiers: HGNCID 6514 and HGNC ID 6515 respectively. LATS1 is sometimes also referredto in the art as WARTS or wts, and LATS2 is sometimes referred to in theart as KPM. Representative LATS sequences, include, but are not limitedto, the protein sequences available from the National Center forBiotechnology Information protein database with the accession numbersNP_004681.1 (LATS1) and NP_001257448.1 (LATS1) and NP_055387.2 (LATS 2),as shown below.

LATS1: NP_004681.1 (Serine/threonine-protein kinase LATS1 isoform 1,homo sapiens) (SEQ ID NO: 1:)    1mkrsekpegy rqmrpktfpa snytvssrqm lqeireslrn lskpsdaaka ehnmskmste   61dprqvrnppk fgthhkalqe irnsllpfan etnssrstse vnpqmlqdlq aagfdedmvi  121qalqktnnrs ieaaiefisk msyqdprreq maaaaarpin asmkpgnvqq svnrkqswkg  181skeslvpqrh gpplgesvay hsespnsqtd vgrplsgsgi safvqahpsn gqrvnppppp  241qvrsvtpppp prgqtppprg ttppppswep nsqtkrysgn meyvisrisp vppgawqegy  301pppplntspm nppnqgqrgi ssvpvgrqpi imqssskfnf psgrpgmqng tgqtdfmihq  361nvvpagtvnr qppppyplta angqspsalq tggsaapssy tngsipqsmm vpnrnshnme  421lynisvpglq tnwpqsssap aqsspssghe iptwqpnipv rsnsfnnplg nrashsansq  481psattvtait papiqqpvks mrvlkpelqt alapthpswi pqpiqtvqps pfpegtasnv  541tvmppvaeap nyqgppppyp khllhqnpsv ppyesiskps kedqpslpke deseksyenv  601dsgdkekkqi ttspitvrkn kkdeerresr iqsyspqafk ffmeqhvenv lkshqqrlhr  661kkqlenemmr vglsqdaqdq mrkmlcqkes nyirlkrakm dksmfvkikt lgigafgevc  721larkvdtkal yatktlrkkd vllrnqvahv kaerdilaea dnewvvrlyy sfqdkdnlyf  781vmdyipggdm msllirmgif peslarfyia eltcavesvh kmgfihrdik pdnilidrdg  841hikltdfglc tgfrwthdsk yyqsgdhprq dsmdfsnewg dpsscrcgdr lkplerraar  901qhqrclahsl vgtpnyiape vllrtgytql cdwwsvgvil femlvgqppf laqtpletqm  961kvinwqtslh ippqaklspe asdliiklcr gpedrlgkng adeikahpff ktidfssdlr 1021qqsasyipki thptdtsnfd pvdpdklwsd dneeenvndt lngwykngkh pehafyeftf 1081rrffddngyp ynypkpieye yinsqgseqq sdeddqntgs eiknrdlvyvLATS1: serine/threonine-protein kinase LATS1 isoform 2 [Homo sapiens]NCBI Reference Sequence: NP_001257448.1 (SEQ ID NO: 2:)   1mkrsekpegy rqmrpktfpa snytvssrqm lqeiresIrn lskpsdaaka ehnmskmste  61dprqvrnppk fgthhkalqe irnsllpfan etnssrstse vnpqmlqdlq aagfdedmvi 121qalqktnnrs ieaaiefisk msyqdprreq maaaaarpin asmkpgnvqq svnrkqswkg 181skeslvpqrh gpplgesvay hsespnsqtd vgrplsgsgi safvqahpsn gqrvnppppp 241qvrsvtpppp prgqtppprg ttppppswep nsqtkrysgn meyvisrisp vppgawqegy 301ppppintspm nppnqgqrgi ssvpvgrqpi imqssskfnf psgrpgmqng tgqtdfmihq 361nvvpagtvnr qppppyplta angqspsalq tggsaapssy tngsipqsmm vpnrnshnme 421lynisvpglq tnwpqsssap aqsspssghe iptwqpnipv rsnsfnnplg nrashsansq 481psattvtait papiqqpvks mrvlkpelqt alapthpswi pqpiqtvqps pfpegtasnv 541tvmppvaeap nyqgppppyp khllhqnpsv ppyesiskps kedqpslpke deseksyenv 601dsgdkekkqi ttspitvrkn kkdeerresr iqsyspqafk ffmeqhvenv lkshqqr1hr 661kkqlenemmr vkpfkmsifi lnhlfawclfLATS 2: NP_055387.2 serine/threonine-protein kinase LATS2 [Homo sapiens]. ((SEQ ID NO: 3:)    1mrpktfpatt ysgnsrqrlq eireglkqps kssvqglpag pnsdtsldak vlgskdatrq   61qqqmratpkf gpyqkalrei rysllpfane sgtsaaaevn rqmlqelvna gcdqemagra  121Ikqtgsrsie aaleyiskmg yldprneqiv rvikqtspgk glmptpvtrr psfegtgdsf  181asyhqlsgtp yegpsfgadg ptaleemprp yvdylfpgvg phgpghqhqh ppkgygasve  241aagahfplqg ahygrphllv pgeplgygvq rspsfqsktp petggyaslp tkgqggppga  301glafpppaag lyvphphhkq agpaahqlhv lgsrsqvfas dsppqslltp srnslnvdly  361elgstsvqqw paatlarrds lqkpgleapp rahvafrpdc pvpsrtnsfn shqprpgppg  421kaepslpapn tvtavtaahi Ihpyksvrvl rpepqtavgp shpawvpapa papapapapa  481aegldakeeh alalggagaf pldveyggpd rrcppppypk hlllrskseq ydldslcagm  541eqslragpne peggdksrks akgdkggkdk kqiqtspvpv rknsrdeekr esriksyspy  601afkffmeqhv enviktyqqk vnrrlqleqe makaglceae qeqmrkilyq kesnynrlkr  661akmdksmfvk iktlgigafg evclackvdt halyamktlr kkdvinrnqv ahvkaerdil  721aeadnewvvk lyysfqdkds lyfvmdyipg gdmmsllirm evfpehlarf yiaeltlaie  781svhkmgfihr dikpdnilid Idghikltdf glctgfrwth nskyyqkgsh vrqdsmepsd  841lwddvsncrc gdrlktleqr arkqhqrcla hslvgtpnyi apevllrkgy tqlcdwwsvg  901vilfemlvgq ppflaptpte tqlkvinwen tlhipaqvkl speardlitk lccsadhrlg  961rngaddlkah pffsaidfss dirkqpapyv ptishpmdts nfdpvdeesp wndasegstk 1021awdtltspnn khpehafyef tfrrffddng ypfrcpkpsg aeasqaessd lessdlvdqt 1081egcqpvyv

LATS is thought to negatively regulate YAP1 activity. “YAP1” refers tothe yes-associated protein 1, also known as YAP or YAP65, which is aprotein that acts as a transcriptional regulator of genes involved incell proliferation. LATS kinases are serine/threonine protein kinasesthat have been shown to directly phosphorylate YAP which results in itscytoplasmic retention and inactivation. Without phosphorylation by LATS,YAP translocates into the nucleus, forming a complex with a DNA bindingprotein, TEAD, and results in downstream gene expression. (Barry E R &Camargo F D (2013) The Hippo superhighway: signaling crossroadsconverging on the Hippo/Yap pathway in stem cells and development.Current opinion in cell biology 25(2):247-253.; Mo J S, Park H W, & GuanK L (2014) The Hippo signaling pathway in stem cell biology and cancer.EMBO reports 15(6):642-656; Pan D (2010) The hippo signaling pathway indevelopment and cancer. Developmental cell 19(4):491-505.)

The Hippo/YAP pathway is involved in numerous cell types and tissues inmammalian systems, including various cancers. In particular, the Hippopathway is evidently involved in the intestine, stomach and esophagus,pancreas, salivary gland, skin, mammary gland, ovary, prostate, brainand nervous system, bone, chrondrocytes, adipose cells, myocytes, Tlymphocytes, B lymphocytes, myeloid cells, kidney, and lung. See Nishioet al., 2017, Genes to Cells 22:6-31.

LATS1 and LATS2 Inhibition

Compounds of Formula A1 or subformulae thereof, in free form or in saltform are potent inhibitors of LATS1 and/or LATS2.

In a preferred embodiment the compounds of Formula A2 or subformulaethereof, in free form or in salt form are potent inhibitors of LATS1 andLATS2.

The inhibition efficacy of the compounds against LATS1 were assayed bythe LATS1 Biochemical HTRF Assay as described in Example A1 below. Theinhibition efficacy of the compounds of the invention against LATS1(LATS1 IC₅₀ in micromolar) in this assay are reported in Table 1A. Itshould be noted that compounds with IC₅₀ greater than 1 micromolar areconsidered inactive in this assay.

The inhibition efficacy of selected compounds against LATS2 were assayedby the LATS2 Biochemical Caliper Assay as described in Example A3 below.The inhibition efficacy of the compounds of the invention against LATS2(LATS2 IC₅₀ in micromolar) in this assay are also reported in Table 1A.It should be noted that compounds with IC₅₀ greater than 1 micromolarare considered inactive in this assay.

LATS Inhibitors

The invention therefore relates to a compound of Formula A2:

-   or a salt, or stereoisomer thereof, wherein-   X¹ is CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or a 9-membered fused bicyclic heteroaryl    that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule; and-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (ix) —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆ alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, provided that when X¹ is CH, R¹ and R² can be taken together    with the nitrogen atom to which both are bound to form a 4- to    6-membered heterocycloalkyl that can include, as ring members, 1 to    2 additional heteroatoms independently selected from N, O, and S,    wherein the 4- to 6-membered heterocycloalkyl formed by R¹ and R²    taken together with the nitrogen atom to which both are bound is    unsubstituted or substituted by 1 to 3 substituents independently    selected from halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰; R³ is    selected from hydrogen, halogen and C₁₋₆alkyl; and R⁵ is selected    from hydrogen, halogen and —NH-(3- to 8-membered heteroalkyl),    wherein the 3- to 8-membered heteroC₃₋₈alkyl of the —NH-(3- to    8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain    members and is unsubstituted or substituted by R⁰;-   with the proviso that:-   (1) when X¹ is N, ring A is 4-primidinyl or 3-fluoro-4-primidinyl,    R¹ is H or methyl, R³ is H or Cl and R⁵ is H; then R² is not    C₂₋₄alkyl that is substituted with a substituent selected from —NH₂,    C₁₋₆alkylamino or t-butyl-carbamoyl-amino and that is optionally    further substituted with unsubstituted phenyl; and-   (2) when X¹ is N, ring A is indazol-5-yl, R¹, R³ and R⁵ are H; then    R² is not C₄alkyl that is substituted with —NH₂.

Unless specified otherwise, the term “compounds of the presentinvention” refers to compounds of Formula A2 or subformulae thereof, orsalts thereof, as well as all stereoisomers (including diastereoisomersand enantiomers), rotamers, tautomers and isotopically labeled compounds(including deuterium substitutions), as well as inherently formedmoieties.

Various (enumerated) embodiments of the invention are described herein.It will be recognized that features specified in each embodiment may becombined with other specified features to provide further embodiments ofthe present invention. When an embodiment is described as being“according to” a previous embodiment, the previous embodiment includessub-embodiments thereof, for example such that when Embodiment 20 isdescribed as being “according to” embodiments 1 to 19, embodiments 1 to19 includes embodiments 19 and 19A.

Embodiment 1. A compound of Formula A2 or a salt thereof, as describedabove.

Embodiment 2. A compound of Formula A2 according to embodiment 1, or asalt thereof, wherein

-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 2 heteroatoms that are selected from    N, provided that at least one of the nitrogen atom ring member is an    unsubstituted nitrogen (—N═) positioned at the 3- or the 4-position    relative to the linking carbon ring member of the 5-membered    heteroaryl or at the para ring position of the 6-membered    heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   -   wherein “*” represents the point of attachment of ring A to the        remainder of the molecule; and    -   wherein ring A is unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, cyano,        C₁₋₆alkyl, C₁₋₆haloalkyl, —NH₂ and C₃₋₆cycloalkyl.

Embodiment 3. A compound of Formula A2, or a salt thereof, according toembodiment 1, wherein ring A is selected from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl,NH₂, and C₃₋₆cycloalkyl;or from

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 4. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 3, wherein ring A is selected from

which are each unsubstituted or substituted by a substituent selectedfrom halogen, cyano, C₁₋₆alkyl, C₁₋₆ haloalkyl, and —NH₂;or is

which is unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 5. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 3, wherein ring A is selected from

Embodiment 6. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 5, wherein ring A is selected from

Embodiment 7. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 6, wherein ring A is selected from

Embodiment 8. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 7, wherein ring A is

Embodiment 9. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 7, wherein ring A is

Embodiment 10. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 7, wherein ring A is

Embodiment 11. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 7, wherein ring A is

Embodiment 12. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 7, wherein ring A is

Embodiment 13. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 5, wherein ring A is

Embodiment 14. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 5, wherein ring A is

Embodiment 15. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 6, wherein ring A is

Embodiment 16. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 3, wherein ring A is

Embodiment 17. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 4, wherein ring A is

Embodiment 18. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 5, wherein ring A is

Embodiment 18A. A compound of Formula A2, or a salt thereof, accordingto any one of embodiments 1 to 5, wherein ring A is

Embodiment 19. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 3, wherein ring A is selected

from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl,NH₂, and C₃₋₆cycloalkyl;

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 20. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 19, wherein R¹ is selected from hydrogen,methyl and ethyl.

Embodiment 21. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 20, wherein R¹ is methyl.

Embodiment 22. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 20, wherein R¹ is hydrogen.

Embodiment 23. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 22, wherein R² is selected from

-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰; (vi) —C(O)R⁸, wherein        R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that is        unsubstituted or substituted by C₁₋₆alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 24. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 22, wherein

-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (vi) —C(O)R⁸, wherein R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that is        unsubstituted or substituted by C₁₋₆alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;        -   and wherein the C atom of the C₁₋₈alkyl that is            unsubstituted or substituted by 1 to 3 substituents (i)            to (x) that is the point of attachment of R² to the            remainder of the molecule is not a —CH₂— group;-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 25. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 23, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by hydroxyl or —C(O)H;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)—C₁₋₆alkoxy, and        C₁₋₆alkyl that is unsubstituted or substituted by —C(O)OH; and    -   (vi) monocyclic C₃₋₆ycloalkyl that is unsubstituted or        substituted by hydroxyl; and-   (b) C₃₋₆ycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl or —C(O)—C₁₋₆alkoxy.

Embodiment 26. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 25, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) C₁₋₆haloalkyl;    -   (ii) —OR⁶, wherein R⁶ is selected from hydrogen, and C₁₋₆alkyl        that is unsubstituted or substituted by hydroxyl; and    -   (iii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by hydroxyl; and-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl.

Embodiment 27. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, wherein R² is C₁₋₈-alkyl that isunsubstituted or substituted by 1 to 2 substituent independentlyselected from C₁₋₆haloalkyl and —OR⁶, wherein R⁶ is selected fromhydrogen, and C₁₋₆alkyl that is unsubstituted or substituted byhydroxyl.

Embodiment 28. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, wherein R² is C₁₋₈-alkyl that isunsubstituted or substituted by hydroxyl.

Embodiment 29. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by C₁₋₆ haloalkyl.

Embodiment 30. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by —O—C₁₋₆ alkyl-OH.

Embodiment 31. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, wherein R² is C₃₋₆cycloalkyl that isunsubstituted or substituted by a substituent selected fromC₁₋₆haloalkyl, C₁₋₆alkylamino, R⁰, and C₁₋₆alkyl that is unsubstitutedor substituted by hydroxyl.

Embodiment 32. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26 and 31, wherein R² is unsubstitutedC₃₋₆cycloalkyl.

Embodiment 33. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26 and 31, wherein R² is C₃₋₆cycloalkyl thatis substituted by C₁₋₆alkyl or C₁₋₆haloalkyl.

Embodiment 34. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 25, wherein R² is selected from isopropyl,s-butyl, t-butyl, 2-methyl-but-2-yl, 2,4,4-trimethylpentan-2-yl,

wherein “*” represents the point of attachment of R² to the remainder ofthe molecule.

Embodiment 34A. A compound of Formula A2, or a salt thereof, accordingto any one of embodiments 1 to 25, wherein R² is selected from

Embodiment 35. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 23 and 25 to 27, wherein R² is selected fromn-propyl, isopropyl, t-butyl,

Embodiment 36. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, 34 and 35, wherein R² is selected from

Embodiment 37. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, 29, and 34 to 36 wherein R² is

Embodiment 38. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, 30, and 34 to 36 wherein R² is

Embodiment 39. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 27, 31, and 34 to 36 wherein R² is

Embodiment 40. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, 31, 34 and 35 wherein R² is selectedfrom

Embodiment 41. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, 31, 33 to 35 and 40, wherein R² isselected from

Embodiment 42. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, 31, 33 to 35 and 40, wherein R² is

Embodiment 43. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 26, 31, 32, 34, 35 and 40 wherein R² is

Embodiment 44. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 23, 25 to 30, and 35 wherein R² is selectedfrom n-propyl, isopropyl and t-butyl.

Embodiment 45. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 23, 25 to 30, 35, and 44 wherein R² isn-propyl.

Embodiment 46. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 30, 35, and 44, wherein R² is isopropyl.

Embodiment 47. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 30, 35, and 44, wherein R² is t-butyl.

Embodiment 48. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 19, wherein

-   X¹ is CH; and-   R¹ and R² taken together with the nitrogen atom to which both are    bound to form a 4- to 6-membered heterocycloalkyl that can include,    as ring members, 1 to 2 additional heteroatoms independently    selected from N, O, and S, wherein the 4- to 6-membered    heterocycloalkyl formed by R¹ and R² taken together with the    nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰.

Embodiment 49. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 19, and 48 wherein

-   X¹ is CH; and-   R¹ and R² are taken together with the nitrogen atom to which both    are bound to form a 5- or 6-membered heterocycloalkyl that can    include, as ring member, 1 to 2 additional heteroatom selected from    N, O and S, wherein the 5- or 6-membered heterocycloalkyl formed by    R¹ and R² taken together with the nitrogen atom to which both are    bound is unsubstituted or substituted by 1 to 3 substituents    independently selected from hydroxyl, C₁₋₄alkyl and C₁₋₄haloalkyl.

Embodiment 50. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 19, 48 and 49 wherein

-   X¹ is CH; and-   R¹ and R² are taken together with the nitrogen atom to which both    are bound to form a 6-membered heterocycloalkyl that can include, as    ring member, an additional heteroatom selected from N and O, wherein    the 6-membered heterocycloalkyl formed by R¹ and R² taken together    with the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    hydroxyl, C₁₋₆alkyl and C₁₋₆haloalkyl.

Embodiment 51. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 19, and 48 to 50 wherein

-   X¹ is CH; and-   R¹ and R² are taken together with the nitrogen atom to which both    are bound to form a 6-membered heterocycloalkyl selected from    piperidinyl, piperazinyl and morpholinyl, wherein the piperidinyl,    piperazinyl or morpholinyl is unsubstituted or substituted by 1 to 3    substituents independently selected from hydroxyl and C₁₋₆alkyl.

Embodiment 52. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 51, wherein R³ is selected from hydrogen,chloro and methyl.

Embodiment 53. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 52, wherein R³ is hydrogen.

Embodiment 53A. A compound of Formula A2, or a salt thereof, accordingto any one of embodiments 1 to 52, wherein R³ is chloro.

Embodiment 53B. A compound of Formula A2, or a salt thereof, accordingto any one of embodiments 1 to 52, wherein R³ is methyl.

Embodiment 54. A compound of the Formula A2, or a salt thereof,according to any one of embodiments 1 to 53, wherein R⁵ is selected fromhydrogen and chloro.

Embodiment 55. A compound of Formula A2, or a salt thereof, according toany one of embodiments 1 to 54, wherein R⁵ is hydrogen.

Embodiment 56. A compound of Formula A2, or a salt thereof, according toembodiment 1, wherein the compound is of Formula A3:

-   wherein-   X¹ is CH or N;-   Ring A is

each of which is unsubstituted or substituted by a substituent selectedfrom halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and —NH₂;

-   R¹ is hydrogen or unsubstituted C₁₋₆alkyl; and-   R² is-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 substituents    selected from    -   (i) C₁₋₄haloalkyl and    -   (ii) —OR⁶, wherein R⁶ is selected from hydrogen and C₁₋₆alkyl        that is unsubstituted or substituted by hydroxyl; or-   (b) monocyclic C₃₋₆cycloalkyl that is unsubstituted or substituted    by C₁₋₆alkyl or C₁₋₆ haloalkyl.

Embodiment 57. A compound of Formula A2, or a salt thereof, according toembodiment 56, wherein ring A is

Embodiment 58. A compound of Formula A2, or a salt thereof, according toembodiment 56 or embodiment 57, wherein R² is

Embodiment 59. A compound of Formula A2, or a salt thereof, according toembodiment 56 or embodiment 57, wherein R² is n-propyl or tert-butylthat is unsubstituted or substituted by trifluromethyl.

Embodiment 60. A compound of the Formula A2, or a salt thereof,according to embodiment 1, selected from:N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N,N-diethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-butyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;N-ethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-cyclohexylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methyloxetan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;N-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methyl-4-phenylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methanesulfonyl-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)aceticacid;(1R,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-(1-methanesulfonyl-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(2S)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-[(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)amino]aceticacid;(2R)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; methyl2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoate;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-(2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethoxy)ethan-1-ol;2-(hydroxymethyl)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-methyl-3-(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanamido)butanoicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-3-phenylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-{[4-(dimethylamino)oxan-4-yl]methyl}-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-(2-methanesulfonylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(adamantan-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propanamide;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propane-2-sulfonamide;2-(pyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4,4,4-trifluoro-2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentyl)methanol;N-(3-methoxycyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1R,2R)-1-N,2-N-dimethyl-1-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]cyclohexane-1,2-diamine;methyl(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;ethyl1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;N-(butan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbut-3-yn-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1r,3s)-3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutan-1-ol;2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(pyridin-4-yl)-N-(2,4,4-trimethylpentan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(pentan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(tert-butoxy)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4,4,4-trifluoro-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-pentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-[1-(1H-indol-3-yl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(4-fluorophenyl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-phenylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(4-fluorophenyl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; 2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethan-1-ol;N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;1-({[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}methyl)cyclopentan-1-ol;N,N-dimethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxyphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-phenyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;6-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}hexanoic acid;N-(3-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoic acid;N-(1-phenylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butylN-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)carbamate;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;methyl2-(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)acetate;N-(2-methylpropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanenitrile;N-(6-aminohexyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-aminobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanenitrile;N-[2-methyl-1-(2-methylpiperidin-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-[4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl]pyridin-2-amine;2-[1-(benzenesulfonyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-N-tert-butylpyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-methylpyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)pentan-2-ol;N-ethyl-2-(3-fluoropyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-[2-methyl-2-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)propoxy]propan-2-ol;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1-methoxy-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;2-(3-chloropyridin-4-yl)-N-cyclopentylpyrido[3,4-d]pyrimidin-4-amine;1-(2-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2-methylpropoxy)-2-methylpropan-2-ol;N-methyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(4-methanesulfonyl-2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[2-chloro-5-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridin-2-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(1H-indazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3,5-dimethyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1,1,1-trifluoro-2-methylpropan-2-yl)-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(4-hydroxy-2,4-dimethylpentan-2-yl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-(3,5-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(2,3-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-thiazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-[2-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-2-carbonitrile;N-(1-methylcyclopropyl)-2-(1,2-oxazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(dimethyl-1,2-oxazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;N-propyl-2-{7H-pyrrolo[2,3-d]pyrimidin-5-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-cyclopropyl-1H-pyrazol-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-[(1R)-1-phenylethyl]-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(5-methyl-1H-pyrazol-4-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridazin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-oxazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-1,2,3-triazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1,2-oxazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(2H-1,2,3,4-tetrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclobutyl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1H-pyrazol-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-4-yl)-N-(2-methylpropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-amino-2-methylpropan-2-yl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-{[4-(tert-butylamino)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-5-yl]amino}ethoxy)ethan-1-ol;N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[2-methyl-1-(propan-2-yloxy)propan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2S)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2R)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butan-1-ol;N-tert-butyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2,2-dimethyl-1-[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]piperidin-4-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}pentan-2-ol;N-cyclopentyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butyl)amine;N,N-diethyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}propoxy)propan-2-ol;N-propyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N-tert-butyl-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyridazin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyridin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N,N-diethyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;(3-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-3-methylbutyl)dimethylamine;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-4-(piperidin-1-yl)-1,7-naphthyridine;2-(3-fluoropyridin-4-yl)-4-(morpholin-4-yl)-1,7-naphthyridine;N-tert-butyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-N-(2-methylbutan-2-yl)-1,7-naphthyridin-4-amine;2-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-2-methylpropan-1-ol;1-[2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-yl]-2,2-dimethylpiperidin-4-ol;2-(3-fluoropyridin-4-yl)-N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-1,7-naphthyridin-4-amine;4-(4-methylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;4-(piperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;4-(2-methylpiperidin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclobutyl)-1,7-naphthyridin-4-amine;2-methyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine;N-(oxetan-3-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methylcyclopropyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;4-(3,3-dimethylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2,2-dimethyl-4-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)morpholine;N-(1-methylcyclobutyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2,2-dimethyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine;N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine;4-(2-methylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2-methyl-N¹-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine;(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)-1,7-naphthyridine;N-(tert-butyl)-N-methyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methylcyclobutyl)-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;N¹,N¹,3-trimethyl-N³-(2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N¹,N¹,3-trimethyl-N³-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2-methyl-1-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-2-yl)carbamate;tert-butyl(2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)ethyl)carbamate;2-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)ethane-1,2-diamine;N,N,2-trimethyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;N¹,3-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2,2-dimethyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)carbamate;2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butanamide;(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;(S)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;ethyl2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate;N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;(S)-1,1,1-trifluoro-2-methyl-3-((2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)amino)propan-2-ol;N-tert-butyl-2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-amine;2-(3-chloropyridin-4-yl)-N,N-diethyl-1,7-naphthyridin-4-amine;N-((1R,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(S)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1S,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(R)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1S,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1R,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate;N¹,N¹,N3,2,2-pentamethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;N¹,N¹-diethyl-3-methyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N3-(2-(2-fluoropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N³-(2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N¹,N¹,3-trimethyl-N³-(2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N³-(2-(2-aminopyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;and3-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine.

Embodiment 60A. A compound of the Formula A2, or a salt thereof,according to embodiment 1, selected from:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 60B. A compound of the Formula A2, or a salt thereof,according to embodiment 1, selected from:N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 60C. A compound of the Formula A2, or a salt thereof,according to embodiment 1, wherein the compound isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

Embodiment 61. A compound of Formula I, or a salt thereof

-   wherein-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (ix) —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆ alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆    alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰;-   with the proviso that:-   (1) when ring A is 4-primidinyl or 3-fluoro-4-primidinyl, R¹ is H or    methyl, R³ is H or Cl and R⁵ is H; then R² is not C₂₋₄alkyl that is    substituted with a substituent selected from —NH₂, C₁₋₆alkylamino or    t-butyl-carbamoyl-amino and that and that is optionally further    substituted with unsubstituted phenyl; and-   (2) when ring A is indazol-5-yl, R¹, R³ and R⁵ are H; then R² is not    C₄alkyl that is substituted with —NH₂.

Embodiment 62. A compound of Formula I according to embodiment 61, or asalt thereof, wherein

-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 2 heteroatoms that are selected from    N, provided that at least one of the nitrogen atom ring member is an    unsubstituted nitrogen (—N═) positioned at the 3- or the 5-position    relative to the linking carbon ring member of the 5-membered    heteroaryl or at the para ring position of the 6-membered    heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule and ring A is unsubstituted or substituted    by 1 to 2 substituents independently selected from halogen, cyano,    C₁₋₆alkyl, C₁₋₆haloalkyl, —NH₂ and C₃₋₆cycloalkyl.

Embodiment 63. A compound of Formula I, or a salt thereof, according toembodiment 61 or embodiment 62, wherein ring A is selected from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl,NH₂, and C₃₋₆cycloalkyl;or from

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 64. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 63, wherein ring A is selected from

that are each unsubstituted or substituted by a substituent selectedfrom halogen, cyano, C₁₋₆alkyl, C₁₋₆ haloalkyl, and —NH₂;or is

that is unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 65. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 63, wherein ring A is selected from

Embodiment 66. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 65, wherein ring A is selected from

Embodiment 67. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 66, wherein ring A is selected from

Embodiment 68. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 67, wherein ring A is

Embodiment 69. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 67, wherein ring A is

Embodiment 70. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 67, wherein ring A is

Embodiment 71. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 67, wherein ring A is

Embodiment 72. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 67, wherein ring A is

Embodiment 73. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 65, wherein ring A is

Embodiment 74. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 65, wherein ring A is

Embodiment 75. A compound of Formula I or a salt thereof, according toany one of embodiments 61 to 66, wherein ring A is

Embodiment 76. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 63, wherein ring A is

Embodiment 77. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 64, wherein ring A is

Embodiment 78. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 65, wherein ring A is

Embodiment 78A. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 65, wherein ring A is

Embodiment 79. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 63, wherein ring A is selected from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl,NH₂, and C₃₋₆cycloalkyl;or from

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 80. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 79, wherein R¹ is selected from hydrogen,methyl and ethyl.

Embodiment 81. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 80, wherein R¹ is methyl.

Embodiment 82. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 80, wherein R¹ is hydrogen.

Embodiment 83. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 82, wherein R² is selected from

-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (vi) —C(O)R⁸, wherein R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that        and that is unsubstituted or substituted by C₁₋₆alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;-   (b) C₃₋₆ycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that and that is unsubstituted    or substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 84. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 82, wherein

-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (vi) —C(O)R⁸, wherein R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that        and that is unsubstituted or substituted by C₁₋₈-alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;        -   and wherein the C atom of the C₁₋₈-alkyl that is            unsubstituted or substituted by 1 to 3 substituents (i)            to (x) that is the point of attachment of R² to the            remainder of the molecule is not a —CH₂— group.-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that and that is unsubstituted    or substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 85. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 83, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by hydroxyl or —C(O)H;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)—C₁₋₆alkoxy, and        C₁₋₆alkyl that is unsubstituted or substituted by —C(O)OH; and    -   (vi) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by one hydroxyl; and-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl or —C(O)—C₁₋₆alkoxy.

Embodiment 86. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 85, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) C₁₋₆haloalkyl;    -   (ii) —OR⁶, wherein R⁶ is selected from hydrogen, and C₁₋₆alkyl        that is unsubstituted or substituted by hydroxyl; and    -   (iii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by hydroxyl; and-   (b) C₃₋₆ycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl.

Embodiment 87. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by 1 to 2 substituent independentlyselected from C₁₋₆haloalkyl and —OR⁶, wherein R⁶ is selected fromhydrogen, and C₁₋₆alkyl that is unsubstituted or substituted byhydroxyl.

Embodiment 88. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by hydroxyl.

Embodiment 89. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by C₁₋₆ haloalkyl.

Embodiment 90. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by —O—C₁₋₆ alkyl-OH.

Embodiment 91. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, wherein R² is C₃₋₆cycloalkyl that isunsubstituted or substituted by a substituent selected fromC₁₋₆haloalkyl, C₁₋₆alkylamino, R⁰, and C₁₋₆alkyl that is unsubstitutedor substituted by hydroxyl.

Embodiment 92. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86 and 91, wherein R² is unsubstitutedC₃₋₆cycloalkyl.

Embodiment 93. A compound of the Formula I, or a salt thereof, accordingto any one of embodiments 61 to 86 and 91, wherein R² is C₃₋₆cycloalkylthat is substituted by C₁₋₆alkyl or C₁₋₆haloalkyl.

Embodiment 94. A compound of the Formula I, or a salt thereof, accordingto any one of embodiments 61 to 85, wherein R² is selected fromn-propyl, isopropyl, s-butyl, t-butyl, 2-methyl-but-2-yl,2,4,4-trimethylpentan-2-yl,

wherein “*” represents the point of attachment of R² to the remainder ofthe molecule.

Embodiment 94A. A compound of the Formula I, or a salt thereof, any oneof according to embodiments 61 to 85, wherein R² is selected from

Embodiment 95. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 83 and 85 to 87, wherein R² is selectedfrom n-propyl, isopropyl, t-butyl,

Embodiment 96. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, 94 and 95, wherein R² is selected from

Embodiment 97. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, 89, and 94 to 96, wherein R² is

Embodiment 98. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, 90, and 94 to 96 wherein R² is

Embodiment 99. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 87, 91, and 94 to 96, wherein R² is

Embodiment 100. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, 91, 94 and 95 wherein R² is selectedfrom

Embodiment 101. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, 91, 93 to 95 and 100, wherein R² is

Embodiment 102. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, 91, 93 to 95 and 100, wherein R² is

Embodiment 103. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 86, 91 to 95, 100 and 102, wherein R² is

Embodiment 104. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 83, 85 to 90, and 95, wherein R² isselected from n-propyl, isopropyl and t-butyl.

Embodiment 105. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 83, 85 to 90, 95, and 104, wherein R² isn-propyl.

Embodiment 106. A compound of the Formula I, or a salt thereof,according to any one of embodiments 61 to 90, 95, and 104, wherein R² isisopropyl.

Embodiment 107. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 90, 95, and 104, wherein R² is t-butyl.

Embodiment 108. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 107, wherein R³ is selected from hydrogen,chloro and methyl.

Embodiment 109. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 108, wherein R³ is hydrogen.

Embodiment 109A. A compound of Formula I, or a salt thereof, accordingto any one of embodiments 61 to 108, wherein R³ is chloro.

Embodiment 109B. A compound of Formula I, or a salt thereof, accordingto any one of embodiments 61 to 108, wherein R³ is methyl.

Embodiment 110. A compound of the Formula I, or a salt thereof,according to any one of embodiments 61 to 109, wherein R⁵ is selectedfrom hydrogen, and chloro.

Embodiment 111. A compound of Formula I, or a salt thereof, according toany one of embodiments 61 to 110, wherein R⁵ is hydrogen.

Embodiment 112. A compound of Formula I, or a salt thereof, according toembodiment 61, wherein the compound is of Formula V:

wherein

Ring A is

each of which is unsubstituted or

-   -   substituted by a substituent selected from halogen, C₁₋₆alkyl,        C₁₋₆haloalkyl, and —NH₂;

-   R¹ is hydrogen or unsubstituted C₁₋₆alkyl; and

-   R² is

-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 substituents    selected from    -   (i) C₁₋₄haloalkyl or    -   (ii) —OR⁶, wherein R⁶ is selected from hydrogen and C₁₋₆alkyl        that is unsubstituted or substituted by hydroxyl; or

-   (b) monocyclic C₃₋₆cycloalkyl that is unsubstituted or substituted    by C₁₋₆alkyl or C₁₋₆ haloalkyl.

Embodiment 113. A compound of Formula I, or a salt thereof, according toembodiment 112, wherein ring A is selected from

Embodiment 114. A compound of Formula I, or a salt thereof, according toembodiment 112 or embodiment 113, wherein ring A is

Embodiment 115. A compound of Formula I, or a salt thereof, according toembodiment 112 or embodiment 113, wherein ring A is

Embodiment 116. A compound of Formula I, or a salt thereof, according toany one of embodiments 112 to 115, wherein R² is selected from

Embodiment 116A. A compound of Formula I, or a salt thereof, accordingto any one of embodiments 112 to 115, wherein R² is selected from

Embodiment 117. A compound of Formula I, or a salt thereof, according toany one of embodiments 112 to 116, wherein R² is

Embodiment 118. A compound of Formula I, or a salt thereof, according toany one of embodiments 112 to 116, wherein R² is

Embodiment 119. A compound of Formula I, or a salt thereof, according toany one of embodiments 112 to 116, wherein R² is

Embodiment 120. A compound of the Formula I, or a salt thereof,according to embodiment 61, selected from:N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N,N-diethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-butyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;N-ethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-cyclohexylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methyloxetan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;N-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methyl-4-phenylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methanesulfonyl-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)aceticacid;(1R,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-(1-methanesulfonyl-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(2S)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-[(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)amino]aceticacid;(2R)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; methyl2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoate;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-(2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethoxy)ethan-1-ol;2-(hydroxymethyl)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-methyl-3-(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanamido)butanoicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-3-phenylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-{[4-(dimethylamino)oxan-4-yl]methyl}-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-(2-methanesulfonylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(adamantan-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propanamide;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propane-2-sulfonamide;2-(pyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4,4,4-trifluoro-2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentyl)methanol;N-(3-methoxycyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1R,2R)-1-N,2-N-dimethyl-1-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]cyclohexane-1,2-diamine;methyl(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;ethyl1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;N-(butan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbut-3-yn-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1r,3s)-3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutan-1-ol;2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(pyridin-4-yl)-N-(2,4,4-trimethylpentan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(pentan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(tert-butoxy)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4,4,4-trifluoro-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-pentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-[1-(1H-indol-3-yl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(4-fluorophenyl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-phenylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(4-fluorophenyl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; 2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethan-1-ol;N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;1-({[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}methyl)cyclopentan-1-ol;N,N-dimethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxyphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-phenyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;6-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}hexanoic acid;N-(3-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoic acid;N-(1-phenylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butylN-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)carbamate;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;methyl2-(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)acetate;N-(2-methylpropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanenitrile;N-(6-aminohexyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-aminobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanenitrile;N-[2-methyl-1-(2-methylpiperidin-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-[4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl]pyridin-2-amine;2-[1-(benzenesulfonyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-N-tert-butylpyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-methylpyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)pentan-2-ol;N-ethyl-2-(3-fluoropyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-[2-methyl-2-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)propoxy]propan-2-ol;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1-methoxy-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;2-(3-chloropyridin-4-yl)-N-cyclopentylpyrido[3,4-d]pyrimidin-4-amine;1-(2-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2-methylpropoxy)-2-methylpropan-2-ol;N-methyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(4-methanesulfonyl-2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[2-chloro-5-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridin-2-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(1H-indazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3,5-dimethyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1,1,1-trifluoro-2-methylpropan-2-yl)-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(4-hydroxy-2,4-dimethylpentan-2-yl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-(3,5-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(2,3-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-thiazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-[2-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-2-carbonitrile;N-(1-methylcyclopropyl)-2-(1,2-oxazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(dimethyl-1,2-oxazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;N-propyl-2-{7H-pyrrolo[2,3-d]pyrimidin-5-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-cyclopropyl-1H-pyrazol-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-[(1R)-1-phenylethyl]-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(5-methyl-1H-pyrazol-4-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridazin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-oxazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-1,2,3-triazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1,2-oxazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(2H-1,2,3,4-tetrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclobutyl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1H-pyrazol-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-4-yl)-N-(2-methylpropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-amino-2-methylpropan-2-yl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N¹,N¹,3-trimethyl-N3-(2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2-methyl-1-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-2-yl)carbamate;tert-butyl(2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)ethyl)carbamate;2-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)ethane-1,2-diamine;N,N,2-trimethyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;N¹,3-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2,2-dimethyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)carbamate;2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butanamide;(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;(S)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;ethyl2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate;N¹,N¹,2,2-tetramethyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;(S)-1,1,1-trifluoro-2-methyl-3-((2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)amino)propan-2-ol;5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-{[4-(tert-butylamino)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-5-yl]amino}ethoxy)ethan-1-ol;N-((1R,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(S)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1S,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(R)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1S,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1R,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate;N¹,N¹,N3,2,2-pentamethyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;N¹,N¹-diethyl-3-methyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N3-(2-(2-fluoropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N3-(2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N¹,N¹,3-trimethyl-N3-(2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N3-(2-(2-aminopyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;and3-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine.

Embodiment 120A. A compound of the Formula I, or a salt thereof,according to embodiment 61, selected from:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 120B. A compound of the Formula I, or a salt thereof,according to embodiment 61, wherein the compound isN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 121. A compound of Formula III, or a salt thereof,

-   wherein-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (ix) —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆ alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;    or-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 122. A compound of formula II according to embodiment 121, ora salt thereof, wherein

-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 2 heteroatoms that are selected from    N, provided that at least one of the nitrogen atom ring member is an    unsubstituted nitrogen (—N═) positioned at the 3- or the 4-position    relative to the linking carbon ring member of the 5-membered    heteroaryl or at the para ring position of the 6-membered    heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule and that is unsubstituted or substituted    by 1 to 2 substituents independently selected from halogen, cyano,    C₁₋₆alkyl, C₁₋₆haloalkyl, —NH₂ and C₃₋₆cycloalkyl.

Embodiment 123. A compound of Formula II, or a salt thereof, accordingto embodiment 121 or embodiment 122, wherein ring A is selected

from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆ haloalkyl,NH₂, and C₃₋₆cycloalkyl;or from

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 124. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 123, wherein ring A is selected

from

which are each unsubstituted or substituted by a substituent selectedfrom halogen, cyano, C₁₋₆alkyl, C₁₋₆haloalkyl, and —NH₂;or is

which is unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 125. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 123, wherein ring A is selected from

Embodiment 126. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 125, wherein ring A is selected from

Embodiment 127. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 126, wherein ring A is selected from

Embodiment 128. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 127, wherein ring A is

Embodiment 129. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 127, wherein ring A is

Embodiment 130. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 127, wherein ring A is

Embodiment 131. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 127, wherein ring A is

Embodiment 132. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 127, wherein ring A is

Embodiment 133. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 125, wherein ring A is

Embodiment 134. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 125, wherein ring A is

Embodiment 135. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 126, wherein ring A is

Embodiment 136. A compound of Formula II, or a salt thereof, accordingto any one of CH₃ embodiments 121 to 123, wherein ring A is

Embodiment 137. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 124, wherein ring A is

Embodiment 138. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 125, wherein ring A is

Embodiment 139. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 123, wherein ring A is selected

from

which are each unsubstituted or substituted by 1 to 2 substituentsindependently selected from cyano, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl,NH₂, and C₃₋₆cycloalkyl;or from

which are each unsubstituted or substituted by C₁₋₆alkyl.

Embodiment 140. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 139, wherein R¹ is selected fromhydrogen, methyl and ethyl.

Embodiment 141. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 140, wherein R¹ is methyl.

Embodiment 142. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 140, wherein R¹ is hydrogen.

Embodiment 143. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 142, wherein

-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (vi) —C(O)R⁸, wherein R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that is        unsubstituted or substituted by C₁₋₆alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 144. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 142, wherein

-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (vi) —C(O)R⁸, wherein R⁸ is R⁰;    -   (vii) —S(O)₂C₁₋₆alkyl;    -   (viii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a substituent selected from C₁₋₆alkyl,        hydroxyC₁₋₆alkyl and R⁰;    -   (ix) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N and O and that is        unsubstituted or substituted by C₁₋₆alkyl; and    -   (x) phenyl that is unsubstituted or substituted by halogen;    -   wherein the C atom of the C₁₋₆alkyl that is unsubstituted or        substituted by 1 to 3 substituents (i) to (x) that is the point        of attachment of R² to the remainder of the molecule is not a        —CH₂— group;-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is unsubstituted or    substituted by R⁰ or —C(O)R⁰; and-   (c) 4-membered heterocycloalkyl comprising, as ring member, a    heteroatom selected from N and O and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰.

Embodiment 145. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 143, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) cyano;    -   (ii) C₂alkynyl;    -   (iii) C₁₋₆haloalkyl;    -   (iv) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by hydroxyl or —C(O)H;    -   (v) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and        R^(7b) is selected from hydrogen, —C(O)—C₁₋₆alkoxy, and        C₁₋₆alkyl that is unsubstituted or substituted by —C(O)OH; and    -   (vi) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by a hydroxyl; and-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl or —C(O)—C₁₋₆alkoxy.

Embodiment 146. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 145, wherein

-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from    -   (i) C₁₋₆haloalkyl;    -   (ii) —OR⁶, wherein R⁶ is selected from hydrogen, and C₁₋₆alkyl        that is unsubstituted or substituted by hydroxyl; and    -   (iii) monocyclic C₃₋₆cycloalkyl that is unsubstituted or        substituted by hydroxyl; and-   (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, and C₁₋₆alkyl that is unsubstituted or substituted    by hydroxyl.

Embodiment 147. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 146, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by 1 to 2 substituent independentlyselected from C₁₋₆haloalkyl and —OR⁶, wherein R⁶ is selected fromhydrogen, and C₁₋₈-alkyl that is unsubstituted or substituted byhydroxyl.

Embodiment 148. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by hydroxyl.

Embodiment 149. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by C₁₋₆ haloalkyl.

Embodiment 150. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, wherein R² is C₁₋₆alkyl that isunsubstituted or substituted by —O—C₁₋₆alkyl-OH.

Embodiment 151. A compound of the Formula II, or a salt thereof,according to any one of embodiments 121 to 146, wherein R² isC₃₋₆cycloalkyl that is unsubstituted or substituted by a substituentselected from C₁₋₆haloalkyl, C₁₋₆alkylamino, R⁰, and C₁₋₆alkyl that isunsubstituted or substituted by hydroxyl.

Embodiment 152. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 146 and 151, wherein R² isunsubstituted C₃₋₆cycloalkyl.

Embodiment 153. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 146 and 151, wherein R² isC₃₋₆cycloalkyl that is substituted by C₁₋₆ alkyl or C₁₋₆haloalkyl.

Embodiment 154. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 145, wherein R² is selected fromn-propyl, isopropyl, s-butyl, t-butyl, 2-methyl-but-2-yl,2,4,4-trimethylpentan-2-yl,

wherein “*” represents the point of attachment of R² to the remainder ofthe molecule.

Embodiment 155. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 143 and 145 to 147, wherein R² isselected from n-propyl, isopropyl, t-butyl,

Embodiment 156. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, 154 and 155, wherein R² isselected from

Embodiment 157. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, 149, and 154 to 156, wherein R² is

Embodiment 158. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, 150, and 154 to 156, wherein R² is

Embodiment 159. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 147, 151, and 154 to 156, wherein R² is

Embodiment 160. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 146, 151, 154 and 155, wherein R² isselected from

Embodiment 161. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 146, 151, 153 to 155 and 160, whereinR² is

Embodiment 162. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 136, 151, 153 to 155 and 160, whereinR² is

Embodiment 163. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 136, 151, 152, 154, 155, and 160,wherein R² is

Embodiment 164. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 133, 135 to 150, and 155, wherein R² isselected from n-propyl, isopropyl and t-butyl.

Embodiment 165. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 143, 145 to 150, 155, and 164, whereinR² is n-propyl.

Embodiment 166. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 150, 155, and 164, wherein R² isisopropyl.

Embodiment 167. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 150, 155, and 164, wherein R² ist-butyl.

Embodiment 168. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 139, wherein R¹ and R² taken togetherwith the nitrogen atom to which both are bound to form a 4- to6-membered heterocycloalkyl that can include, as ring members, 1 to 2additional heteroatoms independently selected from N, O, and S, whereinthe 4- to 6-membered heterocycloalkyl formed by R¹ and R² taken togetherwith the nitrogen atom to which both are bound is unsubstituted orsubstituted by 1 to 3 substituents independently selected from halogen,C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰.

Embodiment 169. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 139, and 168, wherein R¹ and R² aretaken together with the nitrogen atom to which both are bound to form a5- or 6-membered heterocycloalkyl that can include, as ring member, 1 to2 additional heteroatom selected from N, O and S, wherein the 5- or6-membered heterocycloalkyl formed by R¹ and R² taken together with thenitrogen atom to which both are bound is unsubstituted or substituted by1 to 3 substituents independently selected from hydroxyl, C₁₋₄alkyl andC₁₋₄haloalkyl.

Embodiment 170. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 139, 168, and 169, wherein R¹ and R²are taken together with the nitrogen atom to which both are bound toform a 6-membered heterocycloalkyl that can include, as ring member, anadditional heteroatom selected from N and O, wherein the 6-memberedheterocycloalkyl formed by R¹ and R² taken together with the nitrogenatom to which both are bound is unsubstituted or substituted by 1 to 3substituents independently selected from hydroxyl, C₁₋₆alkyl andC₁₋₆haloalkyl.

Embodiment 171. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 139, and 168 to 170, wherein R¹ and R²are taken together with the nitrogen atom to which both are bound toform a 6-membered heterocycloalkyl selected from piperidinyl,piperazinyl and morpholinyl, wherein the piperidinyl, piperazinyl ormorpholinyl is unsubstituted or substituted by 1 to 3 substituentsindependently selected from hydroxyl and C₁₋₆alkyl.

Embodiment 172. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 171, wherein R³ is selected fromhydrogen, chloro and methyl.

Embodiment 173. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 172, wherein R³ is hydrogen.

Embodiment 174. A compound of the Formula II, or a salt thereof,according to any one of embodiments 121 to 173, wherein R⁵ is selectedfrom hydrogen, and chloro.

Embodiment 175. A compound of Formula II, or a salt thereof, accordingto any one of embodiments 121 to 174, wherein R⁵ is hydrogen.

Embodiment 176. A compound of the Formula II, or a salt thereof,according to embodiment 121, wherein the compound is of Formula VI:

wherein

-   Ring A is

each unsubstituted or substituted by a substituent selected fromhalogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and —NH₂;

-   R¹ is hydrogen or unsubstituted C₁₋₆alkyl; and-   R² is-   (a) C₁₋₆alkyl that is unsubstituted or substituted by a substituent    selected from    -   (i) di-C₁₋₆alkylamino;    -   (ii) C₁₋₆haloalkyl;    -   (iii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰; or-   (b) monocyclic C₃₋₆cycloalkyl that is unsubstituted or substituted    by C₁₋₆alkyl or C₁₋₆ haloalkyl;-   or R¹ and R² may be taken together with the nitrogen to which they    are bound to form a 6-membered heterocycloalkyl, which is    unsubstituted or substituted by 1 to 3 substituted selected from    C₁₋₈-alkyl and hydroxyl.

Embodiment 177. A compound of the Formula II, or a salt thereof,according to embodiment 176, wherein ring A is

Embodiment 178. A compound of the Formula II, or a salt thereof,according to embodiment 176 or embodiment 177, wherein R² is selectedfrom ethyl,

Embodiment 179. A compound of the Formula II, or a salt thereof,according to any one of embodiments 176 to 178, wherein R² istert-butyl.

Embodiment 180. A compound of the Formula II, or a salt thereof,according to embodiment 121, selected from:N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[2-methyl-1-(propan-2-yloxy)propan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2S)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2R)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butan-1-ol;N-tert-butyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2,2-dimethyl-1-[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]piperidin-4-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}pentan-2-ol;N-cyclopentyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butyl)amine;N,N-diethyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}propoxy)propan-2-ol;N-propyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N-tert-butyl-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyridazin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyridin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N,N-diethyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;(3-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-3-methylbutyl)dimethylamine;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-4-(piperidin-1-yl)-1,7-naphthyridine;2-(3-fluoropyridin-4-yl)-4-(morpholin-4-yl)-1,7-naphthyridine;N-tert-butyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-N-(2-methylbutan-2-yl)-1,7-naphthyridin-4-amine;2-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-2-methylpropan-1-ol;1-[2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-yl]-2,2-dimethylpiperidin-4-ol;2-(3-fluoropyridin-4-yl)-N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-amine; and2-(3-chloropyridin-4-yl)-N,N-diethyl-1,7-naphthyridin-4-amine.

Embodiment 180a. A compound of the Formula II, or a salt thereof,according to embodiment 121, wherein the compound isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

Embodiment 181. A compound of Formula A1, or a salt thereof,

-   for use in ocular diseases or disorders, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein        R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 182. A compound of Formula A1 or a salt thereof, for use inocular diseases or disorders according to embodiment 181, wherein thecompound is of the formula selected from Formulae I to IV:

Embodiment 183. A compound of Formula A1 or a salt thereof, for use inocular diseases or disorders according to embodiment 181, wherein thecompound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 184. A compound of the Formula A1 or a salt thereof, for usein ocular diseases or disorders according to embodiment 181, wherein thecompound is according to any one of embodiments 1 to 180.

Embodiment 185. Use of a compound of the Formula A1, or a salt thereof,

In a method of generating an expanded population of limbal stem cells,preferably ex vivo, wherein

-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein        R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 186. Use of a compound of the Formula A1, or a salt thereof,

-   in a method of generating an expanded corneal endothelial cell    population, preferably ex vivo, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein        R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆ alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 187. Use of a compound of the Formula A1 or a salt thereof,according to embodiment 185 or 186, wherein the compound is of theformula selected from Formulae I to IV:

Embodiment 188. Use of a compound of Formula A1 or a salt thereof,according to embodiment 185 or 186, wherein the compound is selectedfrom3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; and2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.

Embodiment 188A. Use of a compound of the Formula A1, or a salt thereof,according to embodiment 185 or 186, wherein the compound is selectedfrom:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amineandN-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 188B. Use of a compound of the Formula A1, or a salt thereof,according to embodiment 185 or 186, wherein the compound is selectedfrom: N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 188C. Use of a compound of the Formula A1, or a salt thereof,according to embodiment 185 or 186, wherein the compound is compound isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

Embodiment 189. Use of a compound of Formula A1 or a salt thereof,according to embodiment 185 or 186, wherein the compound is according toany one of embodiments 1 to 180.

Embodiment 190. A method of treatment of an ocular disease or disordercomprising administering to a subject in need thereof a cell population,wherein the cell population has been grown in the presence of a compoundof Formula A1, or a salt thereof,

-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   (i) halogen;-   (ii) cyano;-   (iii) oxo;-   (iv) C₂alkenyl;-   (v) C₂alkynyl;-   (vi) C₁₋₆haloalkyl;-   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and    R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is    unsubstituted or substituted by —C(O)R⁰;-   (ix) —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   (x) —S(O)₂C₁₋₆alkyl;-   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that    are each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1 to    2 heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   (xiii) phenyl that is unsubstituted or substituted by halogen;-   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring    members, 1 to 4 heteroatoms independently selected from N and O; and-   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰; R³ is selected from    hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 190A. A method of treatment of an ocular disease or disordercomprising administering to a subject in need thereof a limbal stem cellpopulation, wherein said population has been grown in the presence of acompound of Formula A1, or a salt thereof,

-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein        R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₈-alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;    -   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 191. A method of treatment of an ocular disease or disordercomprising administering to a subject in need thereof a cornealendothelial cell population, wherein the population has been grown inthe presence of a compound of Formula A1, or a salt thereof,

-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   (i) halogen;-   (ii) cyano;-   (iii) oxo;-   (iv) C₂alkenyl;-   (v) C₂alkynyl;-   (vi) C₁₋₆haloalkyl;-   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and    R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is    unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein R⁸ is    R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   (x) —S(O)₂C₁₋₆alkyl;-   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that    are each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1 to    2 heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   (xiii) phenyl that is unsubstituted or substituted by halogen;-   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring    members, 1 to 4 heteroatoms independently selected from N and O; and-   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;    -   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 192. A method of treatment of an ocular disease or disorderaccording to embodiment 190 or 191, wherein the compound is of theformula selected from Formulae I to IV:

Embodiment 193. A method of treatment of an ocular disease or disorderaccording to embodiment 190 or 191, wherein the compound is selectedfrom3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 193A. A method of treatment of an ocular disease or disorderaccording to embodiment 190 or 191, wherein the compound is selectedfrom:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amineandN-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 193B. A method of treatment of an ocular disease or disorderaccording to embodiment 190 or 191, wherein the compound is selectedfrom: N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 193C. A method of treatment of an ocular disease or disorderaccording to embodiment 190 or 191, wherein the compound isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

Embodiment 194. A method of promoting cell proliferation according toembodiment 190 or 191, wherein the compound is according to any one ofembodiments 1 to 180.

Embodiment 195. Use of a compound of Formula A1, or a salt thereof,

-   in the manufacture of a medicament for an ocular disease or    disorder, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino,    C₃₋₆cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from    -   (i) halogen;    -   (ii) cyano;    -   (iii) oxo;    -   (iv) C₂alkenyl;    -   (v) C₂alkynyl;    -   (vi) C₁₋₆haloalkyl;    -   (vii) —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that        is unsubstituted or substituted by R⁰ or —C(O)R⁰;    -   (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl,        and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is        unsubstituted or substituted by —C(O)R⁰;    -   (ix) —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;    -   (x) —S(O)₂C₁₋₆alkyl;    -   (xi) monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl        that are each unsubstituted or substituted by 1 to 2        substituents independently selected from halogen, C₁₋₆ alkyl,        hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and        di-(C₁₋₆ alkyl)amino;    -   (xii) 6-membered heterocycloalkyl comprising, as ring members, 1        to 2 heteroatoms independently selected from N, O and S and that        is unsubstituted or substituted by 1 to 2 substituents        independently selected from hydroxyl, halogen, C₁₋₆alkyl,        C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;    -   (xiii) phenyl that is unsubstituted or substituted by halogen;    -   (xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring        members, 1 to 4 heteroatoms independently selected from N and O;        and    -   (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as        ring member, 1 to 2 heteroatoms independently selected from N        and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆ alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆ alkyl)amino, —C(O)R⁰,    and C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;    -   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 196. Use of a compound of Formula A1 or a salt thereof, inthe manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is of the formulaselected from Formulae I to IV:

Embodiment 197. Use of a compound of Formula A1 or a salt thereof, inthe manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; and2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.

Embodiment 197A. Use of a compound of the Formula A1, or a salt thereof,in the manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is selected from:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amineandN-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 197B. Use of a compound of the Formula A1, or a salt thereof,in the manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is selected from:N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 197C. Use of a compound of the Formula A1, or a salt thereof,in the manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is compound isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine.

Embodiment 198. Use of a compound of Formula A1 or a salt thereof, inthe manufacture of a medicament for an ocular disease or disorderaccording to embodiment 195, wherein the compound is according to anyone of embodiments 1 to 180.

Embodiment 199. A compound of Formula A1, or a pharmaceuticallyacceptable salt thereof,

-   for use in promoting liver regeneration and liver regrowth, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₈alkylamino, and di-(C₁₋₈alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₈alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 200. A compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting liver regeneration andliver regrowth according to embodiment 199, wherein the compound is ofthe formula selected from Formulae I to IV:

Embodiment 201. A compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting liver regeneration andliver regrowth according to embodiment 199, wherein the compound isselected from:

-   2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;-   dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;-   N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;-   N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine:-   4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;    and-   N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine.

Embodiment 202. A compound of the Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting liver regeneration andliver regrowth according to embodiment 199, wherein the compound isaccording to any one of embodiments 1 to 180.

Embodiment 203. Use of a compound of the Formula A1, or apharmaceutically acceptable salt thereof,

-   for promoting liver regeneration and liver regrowth, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₈alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 204. Use of a compound of the Formula A1 or apharmaceutically acceptable salt thereof, for promoting liverregeneration and liver regrowth according to embodiment 203, wherein thecompound is of the formula selected from Formulae I to IV:

Embodiment 205. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for promoting liver regeneration and liverregrowth according to embodiment 203, wherein the compound is selectedfrom:

-   2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;-   dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;-   N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;-   N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;    and-   N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine.

Embodiment 206. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for promoting liver regeneration and liverregrowth according to embodiment 203, wherein the compound is accordingto any one of embodiments 1 to 180.

Embodiment 207. A method of promoting liver regeneration and liverregrowth, comprising administering to a subject in need thereof atherapeutically effective amount of a compound of Formula A1, or apharmaceutically acceptable salt thereof,

-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₈alkylamino, and di-(C₁₋₈alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₈alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the-   3- to 8-membered heteroC₃₋₈alkyl of the —NH-(3- to 8-membered    heteroalkyl) comprises 1 to-   2 oxygen atoms as chain members and is unsubstituted or substituted    by R⁰.

Embodiment 208. A method of promoting liver regeneration and liverregrowth, according to embodiment 207, wherein the compound is of theformula selected from Formulae I to IV:

Embodiment 209. A method of promoting liver regeneration and liverregrowth according to embodiment 207, wherein the compound is selectedfrom:

-   2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;-   dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;-   N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;-   N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;    and-   N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine.

Embodiment 210. A method of promoting liver regeneration and liverregrowth according to embodiment 207, wherein the compound is accordingto any one of embodiments 1 to 180.

Embodiment 211. Use of a compound of Formula A1, or a pharmaceuticallyacceptable salt thereof,

-   in the manufacture of a medicament for promoting liver regeneration    and liver regrowth,-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₈alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 212. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting liver regeneration and liver regrowth according to embodiment211, wherein the compound is of the formula selected from Formulae I toIV:

Embodiment 213. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting liver regeneration and liver regrowth according to embodiment211, wherein the compound is selected from:

-   2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;-   dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;-   N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;-   N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;-   2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   N¹,N¹,3-trimethyl-N3-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;-   2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;-   2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;-   N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;    4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;    and-   N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine.

Embodiment 214. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting liver regeneration and liver regrowth according to embodiment211, wherein the compound is according to any one of embodiments 1 to180.

In another embodiment, the present invention relates to a pharmaceuticalcomposition comprising at least one of the compounds of the presentinvention or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates a pharmaceuticalcomposition comprising at least one of the compounds of the presentinvention or a pharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the present invention relates to a compound ofthe present invention, or a pharmaceutically acceptable salt thereof, ora pharmaceutical composition for use as a medicament.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for use in promoting liver regeneration and liver regrowth,comprising a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the present invention also relates the use of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof for the manufacture of a medicament forpromoting liver regeneration and liver regrowth alone, or optionally incombination with another compound of Formula A1 or subformulae thereof,or a pharmaceutically acceptable salt thereof and/or at least one othertype of therapeutic agent.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth comprising administeringto a subject in need thereof a therapeutically effective amount of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth, particularly fortreatment of insufficient liver regrowth following transplantation ofmarginal grafts; for supporting enhanced regrowth of the remnant livermass following extensive hepatectomy; for regeneration of patients' oflivers following acute liver failure from viral hepatitis, drug-inducedliver injury, autoimmune hepatitis, ischemic- and congestive liverdisease; and for treatment of patients with chronic liver injury andunderlying liver fibrosis, from non-alcoholic steatohepatitis, alcoholicsteatohepatitis, chronic viral hepatitis B and C, hemochromatosis,alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liverfibrosis to enhance both regenerative capacity and accelerate fibrosisresolution.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth comprising administeringto a subject in need thereof a therapeutically effective amount of anagent capable of inhibiting the activity of LATS1 and LATS2 kinases;thereby inducing YAP translocation and driving downstream geneexpression for cell proliferation. In a further embodiment, the agent isa compound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth comprising administeringto a subject in need thereof a therapeutically effective amount of anagent capable of inhibiting the activity of LATS kinases; therebyinducing YAP translocation and driving downstream gene expression forcell proliferation. In a preferred embodiment, the agent is a compoundaccording to any one of embodiments 1 to 180.

In another embodiment, the present invention relates to a method forexpanding a population of liver cells ex vivo which comprises contactingthe liver cells with a compound according to any one of embodiments 1 to180. In a preferred embodiment, the method further comprises geneediting said liver cells. Preferably said gene editing targets a geneinvolved in the host versus graft immune response.

In another embodiment, the present invention relates to a method forexpanding a population of liver progenitor cells ex vivo which comprisescontacting the liver progenitor cells with a compound according to anyone of embodiments 1 to 180. In a preferred embodiment, the methodfurther comprises gene editing said liver progenitor cells. Preferablysaid gene editing targets a gene involved in the host versus graftimmune response.

In another embodiment, the present invention relates to a population ofliver cells obtained by contacting liver cells with a compound accordingto any one of embodiments 1 to 180. In a preferred embodiment, the livercells obtained by contacting liver cells with a compound according toany one of embodiments 1 to 180 have been gene edited. Preferably saidgene editing targets a gene involved in the host versus graft immuneresponse.

In another embodiment, the present invention relates to a population ofliver progenitor cells obtained by contacting liver progenitor cellswith a compound according to any one of embodiments 1 to 180. In apreferred embodiment, the liver progenitor cells obtained by contactingliver progenitor cells with a compound according to any one ofembodiments 1 to 180 have been gene edited. Preferably said gene editingtargets a gene involved in the host versus graft immune response.

Embodiment 215. A compound of Formula A1, or a pharmaceuticallyacceptable salt thereof,

-   for use in promoting wound healing, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 216. A compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting wound healing according toembodiment 215, wherein the compound is of the formula selected fromFormulae I to IV:

Embodiment 217. A compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting wound healing according toembodiment 215, wherein the compound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 218. A compound of the Formula A1 or a pharmaceuticallyacceptable salt thereof, for use in promoting wound healing according toembodiment 215, wherein the compound is according to any one ofembodiments 1 to 180.

Embodiment 219. Use of a compound of the Formula A1, or apharmaceutically acceptable salt thereof,

-   for promoting wound healing, wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 220. Use of a compound of the Formula A1 or apharmaceutically acceptable salt thereof, for promoting wound healingaccording to embodiment 219, wherein the compound is of the formulaselected from Formulae I to IV:

Embodiment 221. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for promoting wound healing according toembodiment 219, wherein the compound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; and2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.

Embodiment 222. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, for promoting wound healing according toembodiment 219, wherein the compound is according to any one ofembodiments 1 to 180.

Embodiment 223. A method of promoting wound healing, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula A1, or a pharmaceutically acceptablesalt thereof,

-   wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or, R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 224. A method of promoting wound healing, according toembodiment 223, wherein the compound is of the formula selected fromFormulae I to IV:

Embodiment 225. A method of promoting wound healing according toembodiment 223, wherein the compound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.

Embodiment 226. A method of promoting wound healing according toembodiment 223, wherein the compound is according to any one ofembodiments 1 to 180.

Embodiment 227. Use of a compound of Formula A1, or a pharmaceuticallyacceptable salt thereof,

-   in the manufacture of a medicament for promoting wound healing,    wherein-   X¹ and X² are each independently CH or N;-   Ring A is-   (a) a 5- or 6-membered monocyclic heteroaryl that is linked to the    remainder of the molecule through a carbon ring member and    comprises, as ring member, 1 to 4 heteroatoms that are independently    selected from N, O and S, provided that at least one of the    heteroatom ring member is an unsubstituted nitrogen (—N═) positioned    at the 3- or the 4-position relative to the linking carbon ring    member of the 5-membered heteroaryl or at the para ring position of    the 6-membered heteroaryl; or-   (b) a 9-membered fused bicyclic heteroaryl that is selected from

-   wherein “*” represents the point of attachment of ring A to the    remainder of the molecule;-   wherein ring A is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, cyano, C₁₋₆alkyl,    C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, C₃₋₆    cycloalkyl, and phenylsulfonyl;-   R⁰ is hydroxyl or C₁₋₆alkoxy;-   R¹ is hydrogen or C₁₋₆alkyl;-   R² is selected from-   (a) C₁₋₆alkyl that is unsubstituted or substituted by 1 to 3    substituents independently selected from-   halogen;-   cyano;-   oxo;-   C₂alkenyl;-   C₂alkynyl;-   C₁₋₆haloalkyl;-   —OR⁶, wherein R⁶ is selected from hydrogen, C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰;-   —NR^(7a)R^(7b), wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b)    is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted    or substituted by —C(O)R⁰;-   —C(O)R⁸, wherein R⁸ is R⁰ or —NH—C₁₋₆alkyl-C(O)R⁰;-   —S(O)₂C₁₋₆alkyl;-   monocyclic C₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are    each unsubstituted or substituted by 1 to 2 substituents    independently selected from halogen, C₁₋₆alkyl, hydroxyC₁₋₆ alkyl,    C₁₋₆haloalkyl, R⁰, —NH₂, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino;-   6-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms independently selected from N, O and S and that is    unsubstituted or substituted by 1 to 2 substituents independently    selected from hydroxyl, halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and    di-(C₁₋₆alkyl)amino;-   phenyl that is unsubstituted or substituted by halogen;-   5- or 6-membered monocyclic heteroaryl comprising, as ring members,    1 to 4 heteroatoms independently selected from N and O; and-   9- or 10-membered fused bicyclic heteroaryl comprising, as ring    member, 1 to 2 heteroatoms independently selected from N and O;-   (b) —S(O)₂C₁₋₆alkyl;-   (c) phenyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from halogen, C₁₋₆alkyl and R⁰;-   (d) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2    substituents independently selected from C₁₋₆haloalkyl, R⁰,    C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that is    unsubstituted or substituted by R⁰ or —C(O)R⁰; and-   (e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2    heteroatoms selected from N, O and S and that is unsubstituted or    substituted by 1 to 2 substituents independently selected from    C₁₋₆haloalkyl, R⁰, C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and    C₁₋₆alkyl that is unsubstituted or substituted by R⁰ or —C(O)R⁰;-   or R¹ and R² can be taken together with the nitrogen atom to which    both are bound to form a 4- to 6-membered heterocycloalkyl that can    include, as ring members, 1 to 2 additional heteroatoms    independently selected from N, O, and S, wherein the 4- to    6-membered heterocycloalkyl formed by R¹ and R² taken together with    the nitrogen atom to which both are bound is unsubstituted or    substituted by 1 to 3 substituents independently selected from    halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰;-   R³ is selected from hydrogen, halogen and C₁₋₆alkyl; and-   R⁵ is selected from hydrogen, halogen and —NH-(3- to 8-membered    heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl of the    —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as    chain members and is unsubstituted or substituted by R⁰.

Embodiment 228. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting wound healing according to embodiment 227, wherein thecompound is of the formula selected from Formulae I to IV:

Embodiment 229. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting wound healing according to embodiment 227, wherein thecompound is selected from3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; and2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.

Embodiment 230. Use of a compound of Formula A1 or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament forpromoting wound healing according to embodiment 227, wherein thecompound is according to any one of embodiments 1 to 180.

In another embodiment, the present invention relates to a compositioncomprising at least one of the compounds of Formula A2 or subformulaethereof or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates a pharmaceuticalcomposition comprising at least one of the compounds of Formula A2 orsubformulae thereof, or a pharmaceutically acceptable salt thereof andat least one pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the present invention relates to a compound ofFormula A2 or subformulae thereof, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition for use as a medicament.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for use in promoting liver regeneration and liver regrowth,comprising a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the present invention also relates the use of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof for the manufacture of a medicament forpromoting liver regeneration and liver regrowth alone, or optionally incombination with another compound of Formula A1 or subformulae thereof,or a pharmaceutically acceptable salt thereof and/or at least one othertype of therapeutic agent.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth comprising administeringto a subject in need thereof a therapeutically effective amount of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the present invention relates to a method forpromoting liver regeneration and liver regrowth, particularly fortreatment of insufficient liver regrowth following transplantation ofmarginal grafts; for supporting enhanced regrowth of the remnant livermass following extensive hepatectomy; for regeneration of patients' oflivers following acute liver failure from viral hepatitis, drug-inducedliver injury, autoimmune hepatitis, ischemic- and congestive liverdisease; and for treatment of patients with chronic liver injury andunderlying liver fibrosis, from non-alcoholic steatohepatitis, alcoholicsteatohepatitis, chronic viral hepatitis B and C, hemochromatosis,alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liverfibrosis to enhance both regenerative capacity and accelerate fibrosisresolution.

In another embodiment, the present invention related to a method forpromoting liver regeneration and liver regrowth comprising administeringto a subject in need thereof a therapeutically effective amount of anagent capable of inhibiting the activity of LATS1 and LATS2 kinases;thereby inducing YAP translocation and driving downstream geneexpression for cell proliferation. In a further embodiment, the agent isa compound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for use in promoting wound healing, comprising a compound ofFormula A1 or subformulae thereof, or a pharmaceutically acceptable saltthereof.

In another embodiment, the present invention also relates to the use ofa compound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof for the manufacture of a medicament forpromoting wound healing alone, or optionally in combination with anothercompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof and/or at least one other type of therapeuticagent.

In another embodiment, the present invention relates to a method ofpromoting wound healing comprising administering to a subject in needthereof a therapeutically effective amount of a compound of Formula A1or subformulae thereof, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a method ofpromoting wound healing comprising treating or ameliorating thesymptomology of burns, acute and chronic skin ulcers, wherein the skinulcers include, but are not limited to vascular ulcers, diabetic ulcers,and pressure ulcers.

In another embodiment, the present invention related to a method ofpromoting wound healing comprising administering to a subject in needthereof a therapeutically effective amount of an agent capable ofinhibiting the activity of LATS1 and LATS2 kinases; thereby inducing YAPtranslocation and driving downstream gene expression for cellproliferation. In a further embodiment, the agent is a compound ofFormula A1 or subformulae thereof, or a pharmaceutically acceptable saltthereof.

In another embodiment, the present invention relates to a method oftreatment of an ocular disease or disorder comprising administering to asubject in need thereof a cell population, wherein the population hasbeen grown in the presence of an agent capable of inhibiting theactivity of LATS1 and LATS2 kinases; thereby inducing YAP translocationand driving downstream gene expression for cell proliferation. In afurther embodiment, the agent is a compound of Formula A1 or subformulaethereof, or a salt thereof.

In another embodiment, the present invention relates to a method oftreatment of an ocular disease or disorder comprising administering to asubject in need thereof a limbal stem cell population, wherein thepopulation has been grown in the presence of an agent capable ofinhibiting the activity of LATS1 and LATS2 kinases; thereby inducing YAPtranslocation and driving downstream gene expression for cellproliferation. In a further embodiment, the agent is a compound ofFormula A1 or subformulae thereof, or a salt thereof.

In another embodiment, the present invention relates to a method oftreatment of an ocular disease or disorder comprising administering to asubject in need thereof a corneal endothelial cell population, whereinthe population has been grown in the presence of an agent capable ofinhibiting the activity of LATS1 and LATS2 kinases; thereby inducing YAPtranslocation and driving downstream gene expression for cellproliferation. In a further embodiment, the agent is a compound ofFormula A1 or subformulae thereof, or a pharmaceutically acceptable saltthereof.

In another embodiment, the present invention relates to a method ofpromoting ocular wound healing comprising administering to an eye of asubject a therapeutically effective amount of a compound of theinvention. In one embodiment, the ocular wound is a corneal wound. Inother embodiments, the ocular wound is an injury or surgical wound.

Definitions

The general terms used hereinbefore and hereinafter preferably havewithin the context of this invention the following meanings, unlessotherwise indicated, where more general terms wherever used may,independently of each other, be replaced by more specific definitions orremain, thus defining more detailed embodiments of the invention.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The term “a,” “an,” “the” and similar terms used in the context of thepresent invention (especially in the context of the claims) are to beconstrued to cover both the singular and plural unless otherwiseindicated herein or clearly contradicted by the context.

As used herein, the term “C₁₋₆alkyl” refers to a straight or branchedhydrocarbon chain radical consisting solely of carbon and hydrogenatoms, containing no unsaturation, having from one to eight carbonatoms, and which is attached to the rest of the molecule by a singlebond. The term “C₁₋₄alkyl” is to be construed accordingly. As usedherein, the term “n-alkyl” refers to straight chain (un-branced) alkylradical as defined herein. Examples of C₁₋₆alkyl include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), —C(CH₃)₂CH₂CH(CH₃)₂ and—C(CH₃)₂CH₃.

As used herein, the term “C₂-alkenyl” refers to a straight or branchedhydrocarbon chain radical group consisting solely of carbon and hydrogenatoms, containing at least one double bond, having from two to sixcarbon atoms, which is attached to the rest of the molecule by a singlebond. The term “C₂₋₄alkenyl” is to be construed accordingly. Examples ofC₂-alkenyl include, but are not limited to, ethenyl, prop-1-enyl,but-1-enyl, pent-1-enyl, pent-4-enyl and penta-1,4-dienyl.

The term “alkylene” refers to a divalent alkyl group. For example, theterm “C₁₋₆alkylene” or “C₁ to C₆ alkylene” refers to a divalent,straight, or branched aliphatic group containing 1 to 6 carbon atoms.Examples of alkylene include, but are not limited to methylene (—CH₂—),ethylene (—CH₂CH₂—), n-propylene (—CH₂CH₂CH₂—), iso-propylene(—CH(CH₃)CH₂—), n-butylene, sec-butylene, iso-butylene, tert-butylene,n-pentylene, isopentylene, neopentylene and n-hexylene.

As used herein, the term “C₂-alkynyl” refers to a straight or branchedhydrocarbon chain radical group consisting solely of carbon and hydrogenatoms, containing at least one triple bond, having from two to sixcarbon atoms, and which is attached to the rest of the molecule by asingle bond. The term “C₂₋₄alkynyl” is to be construed accordingly.Examples of C₂₋₆alkynyl include, but are not limited to, ethynyl,prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl and penta-1,4-diynyl.

As used herein, the term “C₁₋₆alkoxy” refers to a radical of the formula—OR_(a), where R_(a) is a C₁₋₆alkyl radical as generally defined above.“C_(1-s) alkoxy” or “C₁ to C₆ alkoxy” is intended to include C₁, C₂, C₃,C₄, C₅, and C₆ alkoxy groups (that is 1 to 6 carbons in the alkylchain). Examples of C₁₋₆alkoxy include, but are not limited to, methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.

As used herein, the term “C₁₋₆alkylamino” refers to a radical of theformula —NH—R_(a), where R_(a) is a C₁₋₄alkyl radical as defined above.

As used herein, the term “di-(C₁₋₆alkyl)amino” refers to a radical ofthe formula —N(R_(a))—R_(a), where each R_(a) is a C₁₋₄alkyl radical,which may be the same or different, as defined above.

As used herein, the term “cyano” means the radical *—C≡N The term“cycloalkyl” refers to nonaromatic carbocyclic ring that is a fullyhydrogenated ring, including mono-, bi- or poly-cyclic ring systems.“C₃₋₁₀cycloalkyl” or “C₃ to C₁₀ cycloalkyl” is intended to include C₃,C₄, C₅, C₆, C₇, C₈, C₉ and C₁₀ cycloalkyl groups that is 3 to 10 carbonring members). Example cycloalkyl groups include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and norbornyl.

“Fused ring”, as used herein, refers to a multi-ring assembly whereinthe rings comprising the ring assembly are so linked that the ring atomsthat are common to two rings are directly bound to each other. The fusedring assemblies may be saturated, partially saturated, aromatics,carbocyclics, heterocyclics, and the like. Non-exclusive examples ofcommon fused rings include decalin, naphthalene, anthracene,phenanthrene, indole, benzofuran, purine, quinoline, and the like.

“Halogen” refers to bromo, chloro, fluoro or iodo; preferably fluoro,chloro or bromo.

As used herein, the term “haloalkyl” is intended to include bothbranched and straight-chain saturated alkyl groups as defined abovehaving the specified number of carbon atoms, substituted with one ormore halogens. For example, “C₁₋₆haloalkyl” or “C₁ to C₆ haloalkyl” isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl chain. Examples ofhaloalkyl include, but are not limited to, fluoromethyl, difluoromethyl,trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl,2,2,2-trifluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl and1,4,4-trifluorobutan-2-yl, heptafluoropropyl, and heptachloropropyl.

“Heteroalkyl”, as used herein, refers to an alkyl, as defined herein,where one or more of the carbon atoms within the alkyl chain arereplaced by heteroatoms independently selected from N, O and S. InC_(X-Y)hetereoalkyl or x- to y-membered heteroalkyl, as used herein, x-ydescribe the number of chain atoms (carbon and heteroatoms) on theheteroalkyl. For example C₃-sheteroalkyl refers to an alkyl chain with 3to 8 chain atoms. Further, heteroalkyl as defined herein the atomlinking the radical to the remainder of the molecule must be a carbon.Representative example of 3- to 8-membered heteroalkyl include, but arenot limited to —(CH₂)OCH₃, —(CH₂)₂OCH(CH₃)₂, —(CH₂)₂—O—(CH₂)₂—OH and—(CH₂)₂—(O—(CH₂)₂)₂—OH.

The term “heteroaryl” refers to aromatic moieties containing at leastone heteroatom (e.g., oxygen, sulfur, nitrogen or combinations thereof)within a 5- to 10-membered aromatic ring system Examples of heteroarylinclude, but are not limited to pyrrolyl, pyridyl, pyrazolyl, indolyl,indazolyl, thienyl, furanyl, benzofuranyl, oxazolyl, isoxazolyl,imidazolyl, triazolyl, tetrazolyl, triazinyl, pyrimidinyl, pyrazinyl,thiazolyl, purinyl, benzimidazolyl, quinolinyl, isoquinolinyl,quinoxalinyl, benzopyranyl, benzothiophenyl, benzoimidazolyl,benzoxazolyl and 1H-benzo[d][1,2,3]triazolyl. The heteroaromatic moietymay consist of a single or fused ring system. A typical singleheteroaryl ring is a 5- to 6-membered ring containing one to fourheteroatoms independently selected from N, O and S and a typical fusedheteroaryl ring system is a 9- to 10-membered ring system containing oneto four heteroatoms independently selected from N, O and S. The fusedheteroaryl ring system may consist of two heteroaryl rings fusedtogether or a heteroaryl fused to an aryl (e.g., phenyl).

As used herein, the term “heteroatoms” refers to nitrogen (N), oxygen(O) or sulfur (S) atoms. Unless otherwise indicated, any heteroatom withunsatisfied valences is assumed to have hydrogen atoms sufficient tosatisfy the valences, and when the heteroatom is sulfur, it can beunoxidized (S) or oxidized to S(O) or S(O)₂.

The term “hydroxyl” or “hydroxy”, as used herein, refers to the radical—OH.

“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NH—, —S—, —S(O)— and —S(O)₂—, Examplesof 3 to 8 membered heterocycloalkyl include, but are not limited tooxiranyl, aziridinyl, azetidinyl, imidazolidinyl, pyrazolidinyl,tetrahydrofuranyl, tetrahydrothienyl, tetrahydrothienyl 1,1-dioxide,oxazolidinyl, thiazolidinyl, pyrrolidinyl, pyrrolidinyl-2-one,morpholinyl, piperazinyl, piperidinyl, pyrazolidinyl,hexahydropyrimidinyl, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl,thiomorpholinyl, sulfanomorpholinyl, sulfonomorpholinyl andoctahydropyrrolo[3,2-b]pyrrolyl.

The term “oxo”, as used herein, refers to the divalent radical═O.

As referred to herein, the term “substituted” means that at least onehydrogen atom is replaced with a non-hydrogen group, provided thatnormal valencies are maintained and that the substitution results in astable compound. When a substituent is oxo (i.e., ═O), then 2 hydrogenson the atom are replaced. In cases wherein there are nitrogen atoms(e.g., amines) present in compounds of the present invention, these maybe converted to N-oxides by treatment with an oxidizing agent (e.g.,mCPBA and/or hydrogen peroxides) to afford other compounds of theinvention.

As used herein, the term “unsubstituted nitrogen” refers to a nitrogenring atom that has no capacity for substitution due to its linkage toits adjacent ring atoms by a double bond and a single bond (—N═). Forexample, the nitrogen at the para position of the 4-pyridyl

is an “unsubstituted” nitrogen, andthe nitrogen at the 4-position, inreference to the linking C-ring atom, of 1H-pyrazol-4-yl,

is an “unsubstituted” nitrogen.

As a person of ordinary skill in the art would be able to understand,for example, a ketone (—CH—C(═O)—) group in a molecule may tautomerizeto its enol form(—C═C(OH)—). Thus, this invention is intended to coverall possible tautomers even when a structure depicts only one of them.

As used herein,

are symbols denoting the point of attachment of X, to other part of themolecule.

When any variable occurs more than one time in any constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown to be substituted with 0-3R groups, then said group maybe unsubstituted or substituted with up to three R groups, and at eachoccurrence R is selected independently from the definition of R.

Unless specified otherwise, the term “compound of the present invention”or “compounds of the present invention” refers to compounds of FormulaA2 and subformulae thereof, as well as isomers, such as stereoisomers(including diastereoisomers, enantiomers and racemates), geometricalisomers, conformational isomers (including rotamers and astropisomers),tautomers, isotopically labeled compounds (including deuteriumsubstitutions), and inherently formed moieties (e.g., polymorphs,solvates and/or hydrates). When a moiety is present that is capable offorming a salt, then salts are included as well, in particularpharmaceutically acceptable salts.

It will be recognized by those skilled in the art that the compounds ofthe present invention may contain chiral centers and as such may existin different isomeric forms. As used herein, the term “isomers” refersto different compounds that have the same molecular formula but differin arrangement and configuration of the atoms.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term is used to designate a racemic mixture whereappropriate. When designating the stereochemistry for the compounds ofthe present invention, a single stereoisomer with known relative andabsolute configuration of the two chiral centers is designated using theconventional RS system (e.g., (1S,2S)); a single stereoisomer with knownrelative configuration but unknown absolute configuration is designatedwith stars (e.g., (1R*,2R*)); and a racemate with two letters (e.g,(1RS,2RS) as a racemic mixture of (1R,2R) and (1S,2S); (1RS,2SR) as aracemic mixture of (1R,2S) and (1S,2R)). “Diastereoisomers” arestereoisomers that have at least two asymmetric atoms, but which are notmirror-images of each other. The absolute stereochemistry is specifiedaccording to the Cahn-Ingold-Prelog R-S system. When a compound is apure enantiomer the stereochemistry at each chiral carbon may bespecified by either R or S. Resolved compounds whose absoluteconfiguration is unknown can be designated (+) or (−) depending on thedirection (dextro- or levorotatory) which they rotate plane polarizedlight at the wavelength of the sodium D line. Alternatively, theresolved compounds can be defined by the respective retention times forthe corresponding enantiomers/diastereomers via chiral HPLC.

Certain of the compounds described herein contain one or more asymmetriccenters or axes and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-.

Geometric isomers may occur when a compound contains a double bond orsome other feature that gives the molecule a certain amount ofstructural rigidity. If the compound contains a double bond, thesubstituent may be E or Z configuration. If the compound contains adisubstituted cycloalkyl, the cycloalkyl substituent may have a cis- ortrans-configuration.

Conformational isomers (or conformers) are isomers that can differ byrotations about one or more a bonds. Rotamers are conformers that differby rotation about only a single a bond.

The term “atropisomer” refers to a structural isomer based on axial orplanar chirality resulting from restricted rotation in the molecule.

Unless specified otherwise, the compounds of the present invention aremeant to include all such possible isomers, including racemic mixtures,optically pure forms and intermediate mixtures. Optically active (R)-and (S)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques (e.g., separated onchiral SFC or HPLC chromatography columns, such as CHIRALPAK® andCHIRALCEL® available from DAICEL Corp. using the appropriate solvent ormixture of solvents to achieve good separation).

The present compounds can be isolated in optically active or racemicforms. Optically active forms may be prepared by resolution of racemicforms or by synthesis from optically active starting materials. Allprocesses used to prepare compounds of the present invention andintermediates made therein are considered to be part of the presentinvention. When enantiomeric or diastereomeric products are prepared,they may be separated by conventional methods, for example, bychromatography or fractional crystallization.

As used herein, the term “LATS” is the abbreviated name of the largetumor suppressor protein kinase. LATS as used herein refers to LATS1and/or LATS2. LATS1 as used herein refers to the large tumor suppressorkinase 1 and LATS2 refers to the large tumor suppressor kinase 2. LATS1and LATS2 both have serine/threonine protein kinase activity.

As used herein, the term “YAP1” refers to the yes-associated protein 1,also known as YAP or YAP65, which is a protein that acts as atranscriptional regulator of genes involved in cell proliferation.

As used herein, the term “MST1/2” refers to mammalian sterile 20-likekinase-1 and -2.

As used herein, the term “pharmaceutical composition” refers to acompound of the invention, or a pharmaceutically acceptable saltthereof, together with at least one pharmaceutically acceptable carrier.In one embodiment pharmaceutical composition is in a form suitable fortopical, parenteral or injectable administration.

The term “a therapeutically effective amount” of a compound of thepresent invention refers to an amount of the compound of the presentinvention that will elicit the biological or medical response of asubject, for example, reduction or inhibition of an enzyme or a proteinactivity, or ameliorate symptoms, alleviate conditions, slow or delaydisease progression, or prevent a disease, etc. In one non-limitingembodiment, the term “a therapeutically effective amount” refers to theamount of the compound of the present invention that, when administeredto a subject, is effective to (1) at least partially alleviate, inhibit,prevent and/or ameliorate a condition, or a disorder or a disease (i)mediated by LATS activity, or (ii) characterized by activity (normal orabnormal) of LATS; or (2) reduce or inhibit the activity of LATS; or (3)reduce or inhibit the expression of LATS. In another non-limitingembodiment, the term “a therapeutically effective amount” refers to theamount of the compound of the present invention that, when administeredto a cell, or a tissue, or a non-cellular biological material, or amedium, is effective to at least partially reducing or inhibiting theactivity of LATS; or at least partially reducing or inhibiting theexpression of LATS.

The term “subject” includes human and non-human animals. Non-humananimals include vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, cats, horses, cows, chickens, dog, mouse,rat, goat, rabbit, and pig. Preferably, the subject is human. Exceptwhen noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “IC₅₀”, as used herein, refers to the molar concentration of aninhibitor that produces 50% of the inhibition effect.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers to alleviating or ameliorating the disease ordisorder (i.e., slowing or arresting the development of the disease orat least one of the clinical symptoms thereof); or alleviating orameliorating at least one physical parameter or biomarker associatedwith the disease or disorder, including those which may not bediscernible to the patient.

As used herein, the term “prevent”, “preventing” or “prevention” of anydisease or disorder refers to the prophylactic treatment of the diseaseor disorder; or delaying the onset or progression of the disease ordisorder.

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

Depending on the process conditions the compounds of the presentinvention are obtained either in free (neutral) or salt form. Both thefree form and salt form, and particularly “pharmaceutically acceptablesalts” of these compounds are within the scope of the invention.

As used herein, the terms “salt” or “salts” refers to an acid additionor base addition salt of a compound of the invention. “Salts” include inparticular “pharmaceutical acceptable salts”.

The term “pharmaceutically acceptable salts” refers to salts that retainthe biological effectiveness and properties of the compounds of theinvention and, which typically are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example,acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,toluenesulfonic acid, sulfosalicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

In another aspect, the present invention provides compounds of FormulaA2 in acetate, ascorbate, adipate, aspartate, benzoate, besylate,bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate,camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate,citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate,glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide,isethionate, lactate, lactobionate, laurylsulfate, malate, maleate,malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate,napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate,palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,polygalacturonate, propionate, sebacate, stearate, succinate,sulfosalicylate, sulfate, tartrate, tosylate trifenatate,trifluoroacetate or xinafoate salt form.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Isotopes that can be incorporated intocompounds of the invention include, for example, isotopes of hydrogen.

Further, incorporation of certain isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index ortolerability. It is understood that deuterium in this context isregarded as a substituent of a compound of the Formula A1 orsub-formulae thereof. The concentration of deuterium, may be defined bythe isotopic enrichment factor. The term “isotopic enrichment factor” asused herein means the ratio between the isotopic abundance and thenatural abundance of a specified isotope. If a substituent in a compoundof this invention is denoted as being deuterium, such compound has anisotopic enrichment factor for each designated deuterium atom of atleast 3500 (52.5% deuterium incorporation at each designated deuteriumatom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5%deuterium incorporation), at least 5000 (75% deuterium incorporation),at least 5500 (82.5% deuterium incorporation), at least 6000 (90%deuterium incorporation), at least 6333.3 (95% deuterium incorporation),at least 6466.7 (97% deuterium incorporation), at least 6600 (99%deuterium incorporation), or at least 6633.3 (99.5% deuteriumincorporation). It should be understood that the term “isotopicenrichment factor” can be applied to any isotope in the same manner asdescribed for deuterium.

Other examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F³¹P, ³²P, ³⁵S, ³⁶CI, ¹²³I, ¹²⁴I, ¹²⁵I respectively. Accordingly itshould be understood that the invention includes compounds thatincorporate one or more of any of the aforementioned isotopes, includingfor example, radioactive isotopes, such as ³H and ¹⁴C, or those intowhich non-radioactive isotopes, such as ²H and ¹³C are present. Suchisotopically labelled compounds are useful in metabolic studies (with¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detectionor imaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. In particular, an ¹⁸F or labeled compound may be particularlydesirable for PET or SPECT studies. Isotopically-labeled compounds ofFormula A2 can generally be prepared by conventional techniques known tothose skilled in the art or by processes analogous to those described inthe accompanying Examples and Preparations using an appropriateisotopically-labeled reagent in place of the non-labeled reagentpreviously employed.

Any asymmetric atom (e.g., carbon or the like) of the compound(s) of thepresent invention can be present in racemic or enantiomericallyenriched, for example the (R)-, (S)- or (R,S)-configuration. In certainembodiments, each asymmetric atom has at least 50% enantiomeric excess,at least 60% enantiomeric excess, at least 70% enantiomeric excess, atleast 80% enantiomeric excess, at least 90% enantiomeric excess, atleast 95% enantiomeric excess, or at least 99% enantiomeric excess inthe (R)- or (S)-configuration. Substituents at atoms with unsaturateddouble bonds may, if possible, be present in cis-(Z)- or trans-(E)-form.

Accordingly, as used herein a compound of the present invention can bein the form of one of the possible stereoisomers, rotamers,atropisomers, tautomers or mixtures thereof, for example, assubstantially pure geometric (cis or trans) stereoisomers,diastereomers, optical isomers (antipodes), racemates or mixturesthereof.

Any resulting mixtures of stereoisomers can be separated on the basis ofthe physicochemical differences of the constituents, into the pure orsubstantially pure geometric or optical isomers, diastereomers,racemates, for example, by chromatography and/or fractionalcrystallization.

Any resulting racemates of final compounds or intermediates can beresolved into the optical antipodes by known methods, e.g., byseparation of the diastereomeric salts thereof, obtained with anoptically active acid or base, and liberating the optically activeacidic or basic compound. In particular, a basic moiety may thus beemployed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, di-O,O′-p-toluoyl tartaric acid, mandelicacid, malic acid or camphor-10-sulfonic acid. Racemic products can alsobe resolved by chiral chromatography, e.g., high pressure liquidchromatography (HPLC) using a chiral adsorbent.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (e.g., a polypeptide of the invention), which doesnot comprise additions or deletions, for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over aspecified region, or, when not specified, over the entire sequence of areference sequence), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. The invention providespolypeptides or polynucleotides that are substantially identical to thepolypeptides or polynucleotides, respectively, exemplified herein.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide or cell naturally present in aliving animal is not “isolated,” but the same nucleic acid or peptide orcell partially or completely separated from the coexisting materials ofits natural state is “isolated.”

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form.

Unless specifically limited, the term encompasses nucleic acidscontaining known analogues of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

A “cell population” or “population of cells” as used herein comprisescells that proliferate in the presence of a LATS1 and/or LATS2 inhibitorin vivo or ex vivo. In such cells, Hippo signaling typically suppressescell growth, but will proliferate when the pathway is disrupted by LATSinhibition. In certain embodiments, a cell population useful in amethod, preparation, medium, agent, or kit of the invention comprisescells from tissues described above or cells described or providedherein. Such cells include, but are not limited to ocular cells (e.g.,limbal stem cells, corneal endothelial cells), epithelial cells (e.g.,from skin), neural stem cells, mesenchymal stem cells, basal stem cellsof the lungs, embryonic stem cells, adult stem cells, inducedpluripotent stem cells and liver progenitor cells.

Pharmacology and Utility

In one embodiment the present invention relates to small molecule LATSkinase inhibitors for all indications where cell proliferation would befavorable in vivo and/or ex vivo.

Ex vivo cell therapies generally involve expansion of a cell populationisolated from a patient or healthy donor to be transplanted to a patientto establish a transient or stable graft of the expanded cells. Ex vivocell therapies can be used to deliver a gene or biotherapeutic moleculeto a patient, wherein gene transfer or expression of the biotherapeuticmolecule is achieved in the isolated cells. Non-limiting examples of exvivo cell therapies include, but are not limited to, stem celltransplantation (e.g., hematopoietic stem cell transplantation,autologous stem cell transplantation, or cord blood stem celltransplantation), tissue regeneration, cellular immunotherapy, and genetherapy. See for example, Naldini, 2011, Nature Reviews Genetics volume12, pages 301-315.

In one specific aspect the invention relates to small molecule LATSinhibitors, which are capable of activating the YAP pathway to promoteskin cell proliferation, and thereby are useful in promoting woundhealing.

Further examples of the uses of the compounds of the present inventionare inter alia

-   -   1) in the treatment of insufficient liver regrowth following        transplantation of marginal grafts (organs that are felt        inadequate and thus frequently discarded for reasons that        include but not limited to excessive fatty content, small for        body size and older age of donor);    -   2) to support enhanced regrowth of the remnant liver mass        following extensive hepatectomy, for example in the setting of        primary and metastatic tumors of the liver, traumatic liver        injury, and resection of other space occupying lesions of the        liver such as vascular malformations and liver abscesses;    -   3) to promote growth of hepatic cells (e.g. hepatic progenitor        cells (HPCs)) in culture (ex vivo expansion) and possible        subsequent transplantation;    -   4) to regenerate livers of patients with acute liver failure        from viral hepatitis, drug-induced liver injury, autoimmune        hepatitis, ischemic and congestive liver disease; 5) to support        treatment of patients with chronic liver injury and underlying        liver fibrosis, from non-alcoholic steatohepatitis, alcoholic        steatohepatitis, chronic viral hepatitis B and C,        hemochromatosis, alpha-1 anti-trypsin deficiency, Wilson's        disease and drug-induced liver fibrosis to enhance both        regenerative capacity and accelerate fibrosis resolution; or    -   6) to prevent damage and maintain or improve function of organs        ex vivo, with or without perfusion devices.

The YAP/Hippo pathway regulates tissue growth and regeneration in skinand other tissues. The Hippo (MST) kinase cascade, with LATS being theterminal kinases, negatively regulates YAP activity. LATS kinases areserine/threonine protein kinases that have been shown to directlyphosphorylate YAP which results in its cytoplasmic retention andinactivation. Without phosphorylation by LATS, YAP translocates to thenucleus, forming a complex with TEAD and driving the downstream geneexpression for cell proliferation and survival.

The activity of a compound according to the present invention can beassessed by the following in vitro & in vivo methods.

LATS1 and LATS2 Inhibition

Compounds of Formula A1 or subformulae thereof, in free form or inpharmaceutically acceptable salt form are potent inhibitors of LATS1 andLATS2.

The inhibition efficacy of the compounds against LATS1 were assayed bythe LATS1 Biochemical HTRF Assay as described in the Examples Section A.The inhibition efficacy of the compounds of the invention against LATS1(LATS1 IC₅₀ in μM) are reported in Table 1A.

The LATS1 IC₅₀ of the compounds ranged from >10 μM to less than 1 nM,with the majority of the compounds having IC₅₀ below 1 μM. It should benoted that compounds with IC₅₀ greater than 1 μM are considered inactivein this assy.

The inhibition efficacy of selected compounds against LATS2 were assayedby the LATS2 Biochemical Caliper Assay as described in the ExamplesSection A. The inhibition efficacy of the compounds of the inventionagainst LATS2 (LATS2 IC₅₀ in μM) are also reported in Table 1A.

pYAP Suppression (HaCaT Cells)

By the inhibition of LATS1 and LATS2 kinases, compounds of Formula A1suppress phosphorylation of YAP. The ability of the compounds to reducephosphorylation of YAP in human HaCaT cells (a keratinocytes cell line)was measured by the pYAP HTRF Assay as described in the Examples. Theassay results are reported in Table 1C. The data demonstrated thattreatment by compounds of Formula A1 reduced phosphorylation of YAP.

The ability of the compounds of Formula A1 in reducing phosphorylationof YAP can be demonstrated in similar fashion in JHH-5 cells (Fujise etal., Hepatogastroenterology. 1990 October; 37(5):457-60).

YAP Nuclear Translocation (HaCaT Cells)

Without phosphorylation by LATS, YAP translocates to the nucleus. Theeffect of the compounds of Formula A1, or subformulae thereof, on YAPtranslocation in human HaCaT cells were assessed by the YAP NuclearTranslocation Assay as described in the Examples Section A. The resultedEC₅₀ values are reported in Table 1C. The nuclear translocation EC₅₀ranged from >20 μM to 0.3 μM, and selected compounds having EC₅₀ valuesabout 1 μM or below. The data supports that treatment by selectedcompounds of Formula A1 activate the translocation of YAP from thecytoplasma into the nucleus.

The effect of the compounds of Formula A1, or subformulae thereof, onYAP translocation can be demonstrated in similar fashion in JHH-5 cells(Fujise et al., Hepatogastroenterology. 1990 October; 37(5):457-60).

Target Identification

Compounds of Formula A1, or subformulae thereof, selectively targetLATS1/2 versus MST1/2 kinases. To identify the target of the hits fromthe YAP translocation assay screen, the Target Identification Assay asdescribed in the Examples Section A was performed. The results of theassay are described in FIGS. 33A to 33C.

FIG. 33A shows the western blot analyses for pYAP (Ser127) in lysates ofhuman HaCaT cell, treated with vehicle, or transfected with 40 μM ofsiRNA against MST1/2 or LATS1/2, siRNA against LATS1/2 eliminated pYAPsignal whereas siRNA against MST1/2 had minimal impact. It is consistentwith the consensus in the field that LATS is responsible for YAPphosphorylation.

FIG. 33B shows the western blot analysis of cell lysates of human HaCaTcells that were untreated or treated with 9 μM of Example 133 for 1 hourin the presence of 0.5 μM Okadaic acid. pYAP was dramatically inhibited.The result is similar to the study of siRNA against LATS and MSTS (FIG.33A), which suggests Example 133 targets the LATS kinases.

FIG. 33C shows the inhibition of LATS1 activity (relative to DMSOcontrol) in response to concentration of Example 133 ranging from about104 to 1 μM. The data show that Example 133 strongly inhibited LATS1 inthis assay with an IC₅₀ of 1.3 nM. The result further confirmed that thecompounds of the invention are potent inhibitors of the LATS kinases.

Mouse In Vivo Pharmacodynamics (PD): Use in Wound Healing

The compounds of the invention also activate the YAP pathway in vivo. Astudy of YAP pathway target gene expression was conducted in an in vivomouse model as described in the In Vivo PD Assay in the Examples SectionA. Two full-thickness excisional wounds on the dorsum of an anesthetizedmouse were treated topically with vehicle, or 0.2 and 2 mg/mL of Example133. After two daily doses, the skin samples around the wound edge werecollected and Taqman analyses were performed using Cyr61 and Gapdhprobes. mRNA expression levels for the target genes were normalized toGapdh mRNA levels and plotted against the concentration of Example 133in FIG. 34 . The data shows that Example 133 significantly up-regulatedthe YAP target gene Cyr61 when compared to vehicle, and in adose-dependent manner. The result is consistent with known LATS biologyfor activation of the YAP pathway.

In Vivo Skin Cell Proliferation

Further, the compounds of the invention increase skin cell proliferationin vivo when applied topically. The study was conducted as described the“In Vivo Histology and Ki67 Staining Assay” in the Examples Section A.Example 133 or vehicle were applied to intact mouse dorsal skin. Afterthree days of twice-a-day dosing, the skin samples were collected andsubjected to immunohistology staining for Ki67. The sections wereevaluated visually for cell proliferation and for the abundance of Ki67positive cells. The study results are documented in FIGS. 35A and 35B.

FIG. 35A shows representative micrographs of the Ki67 stained mouse skintreated by either vehicle or Example 133. Mouse skin treated by Example133 shows significant increase in basal cell proliferation and epidermalthickness as compared to vehicle. FIG. 35B compares the abundance ofKi67 positive cell in untreated and treated mouse skin. The data shows a10% higher abundance of Ki67 positive cells in skin treated by Example133, which is indicative of a statistically significant induction ofKi67 positive cells. Accordingly, Example 133 significantly increasedskin cell proliferation in mice in vivo. In summary, from high-contentimaging-based phenotypic HTS, compounds of the invention have been shownto be potent LATS inhibitors, which was confirmed by both cellular andbiochemical LATS assays. Also, by using siRNA to knockdown both LATS1and LATS2, LATS as a negative regulator of the YAP pathway has also beenvalidated.

Further, it has been shown, in vitro, that Example 133 activated the YAPpathway, promoted cell proliferation in human keratinocytes (HaCaT).LATS inhibitors induced YAP target gene expression in wounded mouse PDmodels. Topical treatment of Example 133 in mice induced YAP target geneexpression and increased basal layer skin cell growth as evidenced byKi67 staining. Accordingly, Example 133 is useful in promoting skinregeneration.

Compounds of Formula A1 or subformulae thereof, in free form or inpharmaceutically acceptable salt form, therefore may be useful as atherapy for a disease or condition which can benefit from proliferationof cells as described herein. For example, where regeneration of injuredor diseased tissue and organs would benefit a subject, including but notlimited to skin, liver and the cornea. Additionally, the compounds ofFormula A2, or subformulae thereof, may also be used as researchchemicals, e.g. as tool compounds.

Thus, as a further aspect, the present invention provides the use of acompound of Formula A2 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, in therapy.

In a one embodiment, the present invention provides the use of acompound of Formula A1, or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, for treating andameliorating the symptomology of burns, acute skin ulcers and chronicskin ulcers. In one embodiment, the chronic skin ulcer is selected fromvascular, diabetic and pressure ulcers. In a further embodiment, thechronic skin ulcer is selected from venous leg ulcers, diabetic footulcers, and bed sores.

In another aspect, the invention provides a method of treating a diseaseor condition, which is treated by inhibition of LATS kinases, comprisingadministration of a therapeutically acceptable amount of a compound ofFormula A2, or subformulae thereof, or a pharmaceutically acceptablesalt thereof, or a stereoisomer thereof.

In one embodiment, the disease or condition is selected from burns,acute skin ulcer or chronic skin ulcer. In a further embodiment, thechronic skin ulcer is selected from vascular, diabetic, and pressureulcers. In a further embodiment, the chronic skin ulcer is selected fromvenous leg ulcers, diabetic foot ulcers, and bed sores.

Thus, as a further aspect, the present invention provides the use of acompound of Formula A2, or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, for the manufactureof a medicament.

In a further embodiment, the present invention provides the use of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, for the manufactureof a medicament for promoting wound healing, or for the treatment of adisease which may be treated by inhibition of LATS kinases. In anotherembodiment, the disease is burns, acute or chronic skin ulcer. Inanother embodiment, the chronic skin ulcer is selected from diabeticulcers, pressure ulcers and vascular ulcers. In another embodiment, thechronic skin ulcer is selected from venous leg ulcers, diabetic footulcers and bed sores.

Thus, as a further aspect, the present invention provides a compound ofFormula A2 or subformulae thereof, or a pharmaceutically acceptable saltthereof, or a stereoisomer thereof, for use as a medicament.

In a further embodiment, the present invention provides a compound ofFormula A1 or subformulae thereof, or a pharmaceutically acceptable saltthereof, or a stereoisomer thereof, for use as a medicament forpromoting wound healing, or for the treatment of a disease which may betreated by inhibition of LATS kinases. In another embodiment, thedisease is burns, acute skin ulcer or chronic skin ulcer. In anotherembodiment, the chronic skin ulcer is selected from diabetic ulcers,pressure ulcers and vascular ulcers. In another embodiment, the chronicskin ulcer is selected from venous leg ulcers, diabetic foot ulcers andbed sores.

In Vivo Treatment of Mice: Use in Liver

The ability of the compounds of Formula A1 to induce liver regenerationand liver regrowth was measured by treating mice with the compounds asdescribed in the in vivo Assay as described in the Biologic Assaysection infra. The assay results are reported in Table 1C. The resultsshow that the tested compounds reduce pYAP levels in mouse livers whencompared to vehicle control treated mice, indicating LATS kinaseinhibition in vivo in the liver 2 hours post dosing. Increased mRNAexpression of YAP target genes Cyr61 and Ctgf indicates activation ofYAP signaling 2 hours post dosing. Immunohistochemistry staining for theproliferation marker Ki67 further indicates increased liver cellproliferation 24 hours post dosing.

Thus, as a further aspect, the present invention provides the use of acompound of Formula A2 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, in therapy.

In a one embodiment, the present invention provides the use of acompound of Formula A1 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, for promoting liverregeneration and liver regrowth, particularly for the treatment ofinsufficient liver regrowth following transplantation of marginalgrafts; for supporting enhanced regrowth of the remnant liver massfollowing extensive hepatectomy; for regeneration of patients' of liversfollowing acute liver failure from viral hepatitis, drug-induced liverinjury, autoimmune hepatitis, ischemic- and congestive liver disease;and for treatment of patients with chronic liver injury and underlyingliver fibrosis, from non-alcoholic steatohepatitis, alcoholicsteatohepatitis, chronic viral hepatitis B and C, hemochromatosis,alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liverfibrosis to enhance both regenerative capacity and accelerate fibrosisresolution.

In another aspect, the invention provides a method of treating a diseasewhich is treated by inhibition of LATS kinases comprising administrationof a therapeutically acceptable amount of a compound of Formula A1 orsubformulae thereof, or a pharmaceutically acceptable salt thereof, or astereoisomer thereof.

In a further embodiment, the invention provides a method of promotingliver regeneration and liver regrowth, particularly for treatment ofinsufficient liver regrowth following transplantation of marginalgrafts; for supporting enhanced regrowth of the remnant liver massfollowing extensive hepatectomy; for regeneration of patients' of liversfollowing acute liver failure from viral hepatitis, drug-induced liverinjury, autoimmune hepatitis, ischemic- and congestive liver disease;and for treatment of patients with chronic liver injury and underlyingliver fibrosis, from non-alcoholic steatohepatitis, alcoholicsteatohepatitis, chronic viral hepatitis B and C, hemochromatosis,alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liverfibrosis to enhance both regenerative capacity and accelerate fibrosisresolution, comprising administration of a therapeutically acceptableamount of a compound of Formula A1 or subformulae thereof, or apharmaceutically acceptable salt thereof, or a stereoisomer thereof.

Thus, as a further aspect, the present invention provides the use of acompound of Formula A2 or subformulae thereof, or a pharmaceuticallyacceptable salt thereof, or a stereoisomer thereof, for the manufactureof a medicament.

In a further embodiment, the medicament is for promoting liverregeneration and liver regrowth, particularly for treatment ofinsufficient liver regrowth following transplantation of marginalgrafts; for supporting enhanced regrowth of the remnant liver massfollowing extensive hepatectomy; for regeneration of patients' of liversfollowing acute liver failure from viral hepatitis, drug-induced liverinjury, autoimmune hepatitis, ischemic- and congestive liver disease;and for treatment of patients with chronic liver injury and underlyingliver fibrosis, from non-alcoholic steatohepatitis, alcoholicsteatohepatitis, chronic viral hepatitis B and C, hemochromatosis,alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liverfibrosis to enhance both regenerative capacity and accelerate fibrosisresolution

Thus, as a further aspect, the present invention provides a compound ofFormula A2 or subformulae thereof, or a pharmaceutically acceptable saltthereof, or a stereoisomer thereof, for use as a medicament. In afurther embodiment, the medicament is for promoting liver regenerationand liver regrowth, particularly for treatment of insufficient liverregrowth following transplantation of marginal grafts; for supportingenhanced regrowth of the remnant liver mass following extensivehepatectomy; for regeneration of patients' of livers following acuteliver failure from viral hepatitis, drug-induced liver injury,autoimmune hepatitis, ischemic- and congestive liver disease; and fortreatment of patients with chronic liver injury and underlying liverfibrosis, from non-alcoholic steatohepatitis, alcoholic steatohepatitis,chronic viral hepatitis B and C, hemochromatosis, alpha-1 anti-trypsindeficiency, Wilson's disease and drug-induced liver fibrosis to enhanceboth regenerative capacity and accelerate fibrosis resolution.

In another aspect, the present invention provides the use of a compoundof Formula A1 or subformulae thereof, or a salt thereof, or astereoisomer thereof, for promoting growth of hepatic cells in cultureoutside the donor (ex-vivo expansion) or for induction of liverregeneration ex-vivo.

In another aspect, the present invention provides the method forpromoting growth of hepatic cells in culture outside the donor (ex-vivoexpansion) or for induction of liver regeneration ex-vivo using acompound of Formula A1 or subformulae thereof, or a salt thereof, or astereoisomer thereof.

Pharmaceutical Composition and Administration

In another aspect, in embodiments of the invention relating to in vivouse, the present invention provides a pharmaceutical compositioncomprising a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. In afurther embodiment, the composition comprises at least twopharmaceutically acceptable carriers, such as those described herein. Inembodiments of the invention relating to topical uses of the compoundsof the invention, the pharmaceutical composition is formulated in a waythat is suitable for topical administration such as aqueous solutions,suspensions, ointments, creams, gels or sprayable formulations, e.g.,for delivery by aerosol or the like, comprising the active ingredienttogether with one or more of solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives that are known to thoseskilled in the art.

In embodiments of the invention relating to in vivo use, the compound ofthe present invention is typically formulated into pharmaceutical dosageforms to provide an easily controllable dosage of the drug and to givethe patient an elegant and easily handleable product. The dosage regimenfor the compounds of the present invention will, of course, varydepending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. In embodiments of the invention relating to in vivo use,the compounds of the invention may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three, or four times daily.

In embodiments of the invention relating to in vivo use, thepharmaceutical composition or combination of the present invention canbe in unit dosage of about 1-1000 mg of active ingredient(s) for asubject of about 50-70 kg. The therapeutically effective dosage of acompound, the pharmaceutical composition, or the combinations thereof,is dependent on the species of the subject, the body weight, age andindividual condition, the disorder or disease or the severity thereofbeing treated. A physician, clinician or veterinarian of ordinary skillcan readily determine the effective amount of each of the activeingredients necessary to prevent, treat or inhibit the progress of thedisorder or disease.

The compounds of the present invention can be applied topically in theform of aqueous solutions, suspensions, ointments, creams, lotion, gelsor sprayable formulations, e.g., for delivery by aerosol or the like.The dosage may range between about 10⁻³ molar and 10⁻⁹ molarconcentrations. A therapeutically effective amount in vivo may rangedepending on the route of administration, between about 1-100 mg/kg.

In certain instances, it may be advantageous to administer the compoundof the present invention in combination with at least one additionalpharmaceutical (or therapeutic) agent, such as a pain killer, andcombinations thereof. In particular, compositions will either beformulated together as a combination therapeutic or administeredseparately.

In certain instances, it may be advantageous to administer the compoundof the present invention in combination with at least one additionalpharmaceutical (or therapeutic) agent, such as an immunosuppressant forexample corticosteroids, cyclosporine, tacrolimus, and combinations ofimmunosuppressants. In particular, compositions will either beformulated together as a combination therapeutic or administeredseparately.

The compound of the present invention may be administered eithersimultaneously with, or before or after, one or more other therapeuticagent. The compound of the present invention may be administeredseparately, by the same or different route of administration, ortogether in the same pharmaceutical composition as the other agents. Atherapeutic agent is, for example, a chemical compound, peptide,antibody, antibody fragment or nucleic acid, which is therapeuticallyactive or enhances the therapeutic activity when administered to apatient in combination with a compound of the invention.

In one embodiment, the invention provides a product comprising acompound of Formula A1 or subformulae thereof, and at least one othertherapeutic agent as a combined preparation for simultaneous, separateor sequential use in therapy. In one embodiment, the therapy is thetreatment of a disease or condition mediated by LATS1/2. Productsprovided as a combined preparation include a composition comprising thecompound of Formula A1 and the other therapeutic agent(s) together inthe same pharmaceutical composition, or the compound of Formula A1 andthe other therapeutic agent(s) in separate form, e.g. in the form of akit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a compound of Formula A2 or subformulae thereof and anothertherapeutic agent(s). Optionally, the pharmaceutical composition maycomprise a pharmaceutically acceptable carrier, as described above.

In one embodiment related to in vivo use, the invention provides a kitcomprising two or more separate pharmaceutical compositions, at leastone of which contains a compound of Formula A2 or subformulae thereof.In one embodiment, the kit comprises means for separately retaining saidcompositions, such as a container, divided bottle, divided tube, ordivided foil packet. An example of such a kit is a foil packet, astypically used for deliver gel or ointment, and the like.

The kit of an embodiment of the invention related to in vivo use may beused for administering different dosage forms of the active agents, forexample, oral and topical, for administering the separate compositionsat different dosage intervals, or for titrating the separatecompositions against one another. To assist compliance, the kit of theinvention typically comprises directions for administration.

In the combination therapies of the invention, the compound of theinvention and the other therapeutic agent may be manufactured and/orformulated by the same or different manufacturers. Moreover, forembodiments of the invention related to in vivo use, the compound of theinvention and the other therapeutic may be brought together into acombination therapy: (i) prior to release of the combination product tophysicians (e.g. in the case of a kit comprising the compound of theinvention and the other therapeutic agent); (ii) by the physicianthemselves (or under the guidance of the physician) shortly beforeadministration; (iii) in the patient themselves, e.g. during sequentialadministration of the compound of the invention and the othertherapeutic agent.

Accordingly, the invention relates to the use of a compound of FormulaA1 or subformulae thereof for treating a disease or condition mediatedby inhibition of LATS, wherein the medicament is prepared foradministration with another therapeutic agent. The invention alsorelates to the use of another therapeutic agent for treating a diseaseor condition mediated by inhibition of LATS, wherein the medicament isadministered with a compound of Formula A1 or subformulae thereof.

The invention also relates to a compound of formula A1 or subformulaethereof for use in a method of treating a disease or condition mediatedby inhibition of LATS, wherein the compound of Formula A1 or subformulaethereof is prepared for administration with another therapeutic agent.The invention also relates to another therapeutic agent for use in amethod of treating a disease or condition mediated inhibition of LATS,wherein the other therapeutic agent is prepared for administration witha compound of Formula A1 or subformulae thereof.

The invention also relates to a compound of Formula A1 or subformulaethereof for use in a method of treating a disease or condition mediatedby inhibition of LATS, wherein the compound is administered with anothertherapeutic agent. The invention also provides another therapeutic agentfor use in a method of treating a disease or condition mediated byinhibition of LATS, wherein the other therapeutic agent is administeredwith a compound of Formula A1 or subformulae thereof.

The invention also relates to the use of a compound of Formula A1 orsubformulae thereof for treating a disease or condition mediated byLATS1/2, wherein the patient has previously (e.g. within 24 hours) beentreated with another therapeutic agent. The invention also provides theuse of another therapeutic agent for treating a disease or conditionmediated by LATS, wherein the patient has previously (e.g. within 24hours) been treated with a compound of Formula A1 or subformulaethereof.

PREPARATION OF COMPOUNDS

The compounds of the present invention can be prepared in a number ofways known to one skilled in the art of organic synthesis in view of themethods, reaction schemes and examples provided herein. The compounds ofthe present invention can be synthesized using the methods describedbelow, together with synthetic methods known in the art of syntheticorganic chemistry, or by variations thereon as appreciated by thoseskilled in the art. Preferred methods include, but are not limited to,those described below. The reactions are performed in a solvent orsolvent mixture appropriate to the reagents and materials employed andsuitable for the transformations being effected. It will be understoodby those skilled in the art of organic synthesis that the functionalitypresent on the molecule should be consistent with the transformationsproposed. This will sometimes require a judgment to modify the order ofthe synthetic steps or to select one particular process scheme overanother in order to obtain a desired compound of the invention.

The starting materials are generally available from commercial sourcessuch as Aldrich Chemicals (Milwaukee, Wis.) or are readily preparedusing methods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.), Larock,R. C., Comprehensive Organic Transformations, 2^(nd)-ed., Wiley-VCHWeinheim, Germany (1999), or Beilsteins Handbuch der organischen Chemie,4, Aufl. ed. Springer-Verlag, Berlin, including supplements (alsoavailable via the Beilstein online database).

For illustrative purposes, the reaction schemes depicted below providepotential routes for synthesizing the compounds of the present inventionas well as key intermediates. For a more detailed description of theindividual reaction steps, see the Examples section below. Those skilledin the art will appreciate that other synthetic routes may be used tosynthesize the inventive compounds. Although specific starting materialsand reagents are depicted in the schemes and discussed below, otherstarting materials and reagents can be easily substituted to provide avariety of derivatives and/or reaction conditions. In addition, many ofthe compounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

In the preparation of compounds of the present invention, protection ofremote functionality of intermediates may be necessary. The need forsuch protection will vary depending on the nature of the remotefunctionality and the conditions of the preparation methods. The needfor such protection is readily determined by one skilled in the art. Fora general description of protecting groups and their use, see Greene, T.W. et al., Protecting Groups in Organic Synthesis, 4th Ed., Wiley(2007). Protecting groups incorporated in making of the compounds of thepresent invention, such as the trityl protecting group, may be shown asone regioisomer but may also exist as a mixture of regioisomers.

Abbreviations

Abbreviations as used herein, are defined as follows: “1×” for once,“2×” for twice, “3×” for thrice, “° C.” for degrees Celsius, “aq” foraqueous, “Col” for column, “eq” for equivalent or equivalents, “g” forgram or grams, “mg” for milligram or milligrams, “nm” for nanometer ornanometers, “L” for liter or liters, “mL” or “ml” for milliliter ormilliliters, “ul”, “uL”, “μl”, or “μL” for microliter or microliters,“nL” or “nl” for nanoliter or nanoliters, “N” for normal, “uM” or “μM”micromolar, “nM” for nanomolar, “mol” for mole or moles, “mmol” formillimole or millimoles, “min” for minute or minutes, “h” or “hrs” forhour or hours, “RT” for room temperature, “ON” for overnight, “atm” foratmosphere, “psi” for pounds per square inch, “conc.” for concentrate,“aq” for aqueous, “sat” or “sat'd” for saturated, “MW” for molecularweight, “mw” or “pwave” for microwave, “mp” for melting point, “Wt” forweight, “MS” or “Mass Spec” for mass spectrometry, “ESI” forelectrospray ionization mass spectroscopy, “HR” for high resolution,“HRMS” for high resolution mass spectrometry, “LCMS” for liquidchromatography mass spectrometry, “HPLC” for high pressure liquidchromatography, “RP HPLC” for reverse phase HPLC, “TLC” or “tlc” forthin layer chromatography, “NMR” for nuclear magnetic resonancespectroscopy, “nOe” for nuclear Overhauser effect spectroscopy, “1H” forproton, “5 ” for delta, “s” for singlet, “d” for doublet, “t” fortriplet, “q” for quartet, “m” for multiplet, “br” for broad, “Hz” forhertz, “ee” for “enantiomeric excess” and “a”, “p”, “R”, “r”, “S”, “s”,“E”, and “Z” are stereochemical designations familiar to one skilled inthe art.

The following abbreviations used herein below have the correspondingmeanings:

-   -   AC Active Control    -   AIBN azobisisobutyronitrile    -   ATP adenosine triphosphate    -   Bn benzyl    -   Boc tert-butoxy carbonyl    -   Boc₂O di-tert-butyl dicarbonate    -   BSA bovine serum albumin    -   Bu butyl    -   Cs₂CO₃ cesium carbonate anhydrous    -   CHCl₃ chloroform    -   DAST diethylaminosulfurtrifluoride    -   DBU 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine    -   DCM dichloromethane    -   DMAP 4-dimethylaminopyridine    -   DMEM Dulbecco's modified Eagle's medium    -   DMF dimethylformamide    -   DMSO dimethylsulfoxide    -   DPPA diphenylphosphoryl azide    -   DTT dithiolthreitol    -   EA ethyl acetate    -   EDTA ethylenediaminetetraacetic acid    -   Equiv. equivalence    -   Et ethyl    -   Et₂O diethyl ether    -   EtOH ethanol    -   EtOAc ethyl acetate    -   FBS fetal bovine serum    -   HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HCl hydrochloric acid    -   HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   HPMC (hydroxypropyl)methyl cellulose    -   HTRF homogeneous time resolved fluorescence    -   i-Bu isobutyl    -   i-Pr isopropyl    -   KOAc potassium acetate    -   LiAIH₄ lithium aluminium hydride    -   Me methyl    -   mCPBA 3-chloroperoxybenzoic acid    -   MeCN acetonitrile    -   MnO₂ manganese dioxide    -   N₂ nitrogen    -   NaBH₄ sodium borohydride    -   NaHCO₃ sodium bicarbonate    -   Na₂SO₄ sodium sulfate    -   NBS N-Bromosuccinimide    -   NC Neutral Control    -   PBS phosphate buffered saline    -   PFA paraformaldehyde    -   Ph phenyl    -   PPh₃ triphenylphosphine    -   Ph₃P═O triphenylphosphine oxide    -   pYAP phospho-YAP    -   R_(f) retention factor    -   RT room temperature (° C.)    -   Ser serine    -   t-Bu or Bu^(t) tert-butyl    -   T3P® Propane phosphonic acid anhydride    -   TEA triethylamine    -   TFA trifluoroacetic acid    -   TH F tetrahydrofuran    -   UVA Ultraviolet A    -   YAP Yes associated protein (NCBI Gene ID: 10413; official        symbol: (YAP1)

I. General Synthetic Routes

Compounds of Formulae I to VI can be prepared as illustrated in theGeneral Schemes I to III and in greater details in Schemes 1 to 6 below.Detailed description for the synthesis of the intermediates andexemplified compounds are also disclosed below.

General Scheme I for the Preparation of Compounds of Formula I or II

The bicyclic dichloride GS1b could be commercially available when X═C orcould be prepared from aminoisonicotinic acid/amide GS1a throughcyclization and chlorination. The dichloride of GS1b could be aminatedand coupled with the appropriate agents to form GS1c, which furtherfunctionalized to yield Formula I or Formula II through any necessaryfunctionalization, such as but not limited to protection andde-protection steps, reduction, hydrolysis, alkylation, amination,coupling, etc

General Scheme II for the Preparation of Compounds of Formula III

General Scheme III for the Preparation of Compounds of Formula IV

Scheme 1

Compounds of Formula V can be prepared as illustrated in Scheme 1 below.Step C could include amination and any necessary functionalization, suchas but not limited to protection and de-protection steps, reduction,hydrolysis, alkylation, etc.

Scheme 2

Alternatively, compounds of Formula V can be prepared as illustrated inScheme 2. Step C could include amination and any necessaryfunctionalization, such as but not limited to protection andde-protection steps, reduction, hydrolysis, alkylation, etc. Furtherfunctionalization of mono-chloride intermediate 2d by but not limited tometal mediated coupling, amination, alkylation etc. and necessaryprotection and de-protection steps, leads to compounds of Formula V.

Scheme 3

Compounds of Formula I where R⁵ is hydrogen can be prepared asillustrated in Scheme 3. Step C could include amination and anynecessary functionalization, such as but not limited to protection andde-protection steps, reduction, hydrolysis, alkylation, etc. Furtherfunctionalization of mono-chloride intermediate 3d by but not limited tometal mediated coupling, amination, alkylation etc. and necessaryprotection and de-protection steps, leads to compounds of Formula (I)where R⁵ is hydrogen.

Scheme 4

Compounds of Formula I, where R³ and R⁵ are both hydrogen, can beprepared as illustrated in Scheme 4. Step C could include amination andany necessary functionalization, such as but not limited to protectionand de-protection steps, reduction, hydrolysis, alkylation, etc. leadsto compounds of Formula I where R³ and R⁵ are both hydrogen.

Scheme 5

Compounds of Formula I, where R³ is hydrogen, can be prepared asillustrated in Scheme 5. Step D could include amination and anynecessary functionalization, such as but not limited to protection andde-protection steps, reduction, hydrolysis, alkylation, etc. Furtherfunctionalization of mono-chloride intermediate 5d by, but not limitedto, metal mediated coupling, amination, alkylation etc. and necessaryprotection and de-protection steps, leads to compounds of Formula Iwhere R³ is hydrogen,

Scheme 6

Compounds of Formula VI can be prepared from commercially availabledichloride 6a′ (2,4-dichloro-1,7-naphthyridine, Aquila Pharmatech) asillustrated in Scheme 6. Step A could include metal mediated couplingand any necessary functionalization, such as but not limited toprotection and de-protection steps, cyclization, reduction, hydrolysis,alkylation, etc. Step B could include amination and any necessaryfunctionalization, such as but not limited to protection andde-protection steps, reduction, hydrolysis, alkylation, etc.

Preparation of Exemplified Examples

The following Examples have been prepared, isolated and characterizedusing the methods disclosed herein. The following examples demonstrate apartial scope of the invention and are not meant to be limiting of thescope of the invention.

Unless specified otherwise, starting materials are generally availablefrom a non-excluding commercial sources such as TCI Fine Chemicals(Japan), Shanghai Chemhere Co., Ltd. (Shanghai, China), Aurora FineChemicals LLC (San Diego, Calif.), FCH Group (Ukraine), AldrichChemicals Co. (Milwaukee, Wis.), Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn, N.J.), Maybridge Chemical Company, Ltd.(Cornwall, England), Tyger Scientific (Princeton, N.J.), AstraZenecaPharmaceuticals (London, England), Chembridge Corporation (USA), MatrixScientific (USA), Conier Chem & Pharm Co., Ltd (China), Enamine Ltd(Ukraine), Combi-Blocks, Inc. (San Diego, USA), Oakwood Products, Inc.(USA), Apollo Scientific Ltd. (UK), Allichem LLC. (USA) and UkrorgsyntezLtd (Latvia).

LCMS Methods Employed in Characterization of Examples 1-290

Analytical LC/MS is carried out on Agilent systems using ChemStationsoftware. The systems consist of:

-   -   Agilent G1312 Binary Pump    -   Agilent G1367 Well Plate Autosampler    -   Agilent G1316 Thermostated Column Compartment    -   Agilent G1315 Diode Array Detector    -   Agilent 6140/6150 Mass Spectrometer    -   SOFTA Evaporative Light Scattering Detector

Typical method conditions are as follows:

-   -   Flow Rate: 0.9 mL/min    -   Column: 1.8 micrometres 2.1×50 mm Waters Acquity HSS T3 C18        column    -   Mobile Phase A: Water+0.05% TFA    -   Mobile Phase B: Acetonitrile+0.035% TFA    -   Run Time: 2.25 minutes    -   The system runs a gradient from 10% B to 90% B in 1.35 minutes.        A 0.6 minute wash at 100% B follows the gradient. The remaining        duration of the method returns the system to initial conditions.    -   Typical mass spectrometer Scan range is 100 to 1000 amu.

LCMS Methods Employed in Characterization of Examples 291-335

LC-MS (Method 1):

System: Waters Acquity UPLC with Waters SQ detector.

Column: Acquity HSS T3 1.8 μm 2.1×50 mm.

Flow: 1.0 ml/min. Column temperature: 60° C.

Gradient: from 5 to 98% B in 1.4 min, A=water+0.05% formic acid+3.75 mMammonium

acetate, B=acetonitrile+0.04% formic acid.

LC-MS (Method 2):

System: Waters Acquity H-Class UPLC with Waters SQ detector.

Column: BEH C18 1.7 μm 2.1×50 mm

Flow: 3.0 ml/min. Column temperature: 30° C.

Gradient: from 2 to 100% B in 2.7 min, A=2 mM ammoniumacetate/water+0.1% formic acid, B=acetonitrile+0.1% formic acid.

NMR Employed in Characterization of Examples 1-290

Proton spectra are recorded on a Bruker AVANCE II 400 MHz with 5 mm QNPCryoprobe or a Bruker AVANCE III 500 MHz with 5 mm QNP probe unlessotherwise noted. Chemical shifts are reported in ppm relative todimethyl sulfoxide (δ 2.50), chloroform (δ 7.26), methanol (δ 3.34), ordichloromethane (δ 5.32). A small amount of the dry sample (2-5 mg) isdissolved in an appropriate deuterated solvent (1 mL).

NMR Employed in Characterization of Examples 291-335

Proton spectra are recorder on a Bruker Avance 400 NMR spectrometer (400MHz) equipped with a cryo probe or a Bruker Avance 600 NMR spectrometer(600 MHz) equipped with a cryo probe. Chemical shifts (6-values) arereported in ppm downfield from tetramethylsilane, spectra splittingpattern are designated as singlet (s), doublet (d), triplet (t), quartet(q), pentet (p) multiplet, unresolved or more overlapping signals (m),broad signal (br). Solvents are given in parentheses.

Reagents and Materials

Solvents and reagents were purchased from suppliers and used without anyfurther purification. Basic ion exchange resin cartridges PoraPak™ RxnCX 20cc (2 g) were purchased from Waters. Phase separator cartridges(Isolute Phase Separator) were purchased from Biotage. Isolute absorbant(Isolute HM-N) was purchased from Biotage.

ISCO Methods Employed in Purification of Examples

ISCO flash chromatography is carried on Teledyne COMBIFLASH® system withprepacked silica RediSep® column.

Preparative HPLC Methods Employed in Purification of Examples

Preparative HPLC is carried out on Waters Autoprep systems usingMassLynx and FractionLynx software. The systems consist of:

-   -   Waters 2767 Autosampler/Fraction Collector    -   Waters 2525 Binary Pump    -   Waters 515 Makeup pump    -   Waters 2487 Dual Wavelength UV Detector    -   Waters ZQ Mass Spectrometer

Typical Method Conditions are as Follows:

-   -   Flow Rate: 100 mL/min    -   Column: 10 micrometres 19×50 mm Waters Atlantis T3 C18 column    -   Injection Volume: 0-1000 microlitres    -   Mobile Phase A: Water+0.05% TFA    -   Mobile Phase B: Acetonitrile+0.035% TFA    -   Run Time: 4.25 minutes

The system runs a gradient from x % B to y % B as appropriate for theexamples in 3 minutes following a 0.25 minute hold at initialconditions. A 0.5 minute wash at 100% B follows the gradient. Theremaining duration of the method returns the system to initialconditions.

Fraction collection is triggered by mass detection through FractionLynxsoftware.

Chiral Preparative HPLC Methods Employed in Purification of Examples

SFC chiral screening is carried out on a Thar Instruments PrepInvestigator system coupled to a Waters ZQ mass spectrometer. The TharPrep Investigator system consists of:

-   -   Leap HTC PAL autosampler    -   Thar Fluid Delivery Module (0 to 10 mL/min)    -   Thar SFC 10 position column oven    -   Waters 2996 PDA    -   Jasco CD-2095 Chiral Detector    -   Thar Automated Back Pressure Regulator.

All of the Thar components are part of the SuperPure Discovery Seriesline.

The system flows at 2 mL/min (4 mL/min for the WhelkO-1 column) and iskept at 30 degrees C. The system back pressure is set to 125 bar. Eachsample is screened through a battery of six 3 micrometre columns:

-   -   3 micrometre 4.6×50 mm ChiralPak AD    -   3 micrometre 4.6×50 mm ChiralCel OD    -   3 micrometre 4.6×50 mm ChiralCel OJ    -   3 micrometre 4.6×250 mm Whelk 0-1    -   3 micrometre 4.6×50 mm ChiralPak AS    -   3 micrometre 4.6×50 mm Lux-Cellulose-2

The system runs a gradient from 5% co-solvent to 50% co-solvent in 5minutes followed by a 0.5 minute hold at 50% co-solvent, a switch backto 5% co-solvent and a 0.25 minute hold at initial conditions. Inbetween each gradient there is a 4 minute equilibration method the flowsat 5% co-solvent through the next column to be screened. The typicalsolvents screened are MeOH, MeOH+20 mM NH₃, MeOH+0.5% DEA, IPA, andIPA+20 mM NH₃.

Once separation is detected using one of the gradient methods anisocratic method will be developed and, if necessary, scaled up forpurification on the Thar Prep80 system.

Synthesis of Intermediates

Intermediate 1c (Scheme 1):4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine

Step A: In a 20 mL microwave vial was added 3-aminoisonicotinamide (1a,2 g, 14.58 mmol), isonicotinaldehyde (1.521 mL, 16.04 mmol), sodiumbisulfite (1.821 g, 17.50 mmol) and 4-methylbenzenesulfonic acid hydrate(0.277 g, 1.458 mmol) in DMA (Volume: 5 mL) to give an orangesuspension. The reaction was well stirred and heated in microwave at160° C. for 12 min. The reaction mixture was diluted with water andfiltered. The solid was washed with water, MeOH and ether to give 2.03 goff white solid as the product 1b (59%). 1H NMR (400 MHz, DMSO-d6) δ9.18 (d, J=0.9 Hz, 1H), 8.88-8.78 (m, 2H), 8.73 (d, J=5.2 Hz, 1H),8.16-8.08 (m, 2H), 8.03 (dd, J=5.2, 0.9 Hz, 1H).

Step B: In a 5 mL microwave reactor was2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (1b, 500 mg, 2.230 mmol) andphenylphosphonic dichloride (1564 microlitre, 11.15 mmol) to give abrown suspension. The reaction mixture was stirred at 170° C. for 30 minwhen LCMS indicated full conversion. The reaction mixture was quenchedwith ice/water and neutralized with saturated Na₂CO₃, then extractedwith DCM×3 and give the product 1c (74%). 1H NMR (500 MHz, DMSO-d6) δ9.65 (d, J=1.0 Hz, 1H), 8.96 (d, J=5.6 Hz, 1H), 8.87 (s, 2H), 8.47-8.30(m, 2H), 8.18 (dd, J=5.6, 1.0 Hz, 1H). LCMS (m/z [M+H]⁺): 243.1.

Intermediate 2c (Scheme 2): 2,4-dichloropyrido[3,4-d]pyrimidine

Step A: A mixture of urea (40.00 g, 666.00 mmol) and 3-aminoisonicotinicacid (2a, 18.40 g, 133.20 mmol) was heated at 210° C. for 1 hr (NOTE: nosolvent was used). NaOH (2N, 320 mL) was added, and the mixture wasstirred at 90° C. for 1 h. The solid was collected by filtration, andwashed with water. The crude product thus obtained was suspended in HOAc(400 mL), and stirred at 100° C. for 1 h. The mixture was cooled to RT,filtered, and the solid was washed with a large amount of water, andthen dried under the vacuum to givepyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b, 17.00 g, 78% yield)without further purification. LCMS (m/z [M+H]⁺): 164.0.

Step B: To a mixture of pyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b,20.00 g, 122.60 mmol) and POCl₃ (328.03 g, 2.14 mol) in toluene (200 mL)was added DIEA (31.69 g, 245.20 mmol) dropwise and this reaction mixturestirred at 25° C. overnight (18 hr) to give suspension.

The solvent and POCl₃ was removed under vacuum, diluted with DCM (50mL), neutralized with DIEA to pH=7 at −20° C. and concentrated again,the residue was purified by column (20-50% EA/PE) to give the product(2c, 20.00 g, 99.99 mmol, 82% yield) as a yellow solid. 1H NMR (400 MHz,CHLOROFORM-d) δ 9.52 (s, 1H), 8.92 (d, J=5.6 Hz, 1H), 8.04 (d, J=5.6 Hz,1H). LCMS (m/z [M+H]⁺): 200.0.

Intermediate 3c (Scheme 3):4,8-dichloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine

Step A: In a 20 mL microwave reactor was added 3-aminoisonicotinamide(3a, 650 mg, 3.79 mmol), and isonicotinaldehyde (487 mg, 4.55 mmol),sodium bisulfite (788 mg, 7.58 mmol) in DMA (Volume: 10 mL) to give ayellow suspension. The reaction mixture was stirred under microwave at160° C. for 10 min. The reaction mixture was diluted with water,filtered and washed with MeOH and ether. The solid was collected to givethe product 3b (8-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol,300 mg, 29%). 1H NMR (400 MHz, DMSO-d6) δ 8.86-8.80 (m, 2H), 8.45 (d,J=5.1 Hz, 1H), 8.18-8.12 (m, 2H), 8.00 (d, J=5.1 Hz, 1H).

Step B: In a 20 mL microwave reactor was mixed(8-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (3b, 300 mg, 1.160mmol), and phenylphosphonic dichloride (1 mL, 7.13 mmol) to give ayellow suspension. The reaction mixture was stirred under microwave at170° C. for 60 min. The reaction mixture was diluted with water andfiltered. The solid was washed with water, MeOH and ether to give thetitle product 3c (78%). 1H NMR (400 MHz, DMSO-d6) δ 9.05-8.96 (m, 2H),8.52 (d, J=5.1 Hz, 1H), 8.46-8.38 (m, 2H), 8.05 (d, J=5.1 Hz, 1H). (NMRsample was added 1 drop of TFA, otherwise, 2 set of peaks wereobserved). LCMS (m/z [M+H]⁺): 277.0.

Intermediate 4c (Scheme 4, intermediate 4c, wherein X is F and A is4-pyridinyl): 4-chloro-6-fluoro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine

Step A: A solution of isonicotinonitrile (800 mg, 7.69 mmol) in MeOH (30ml) was treated with sodium methoxide (0.474 ml, 5.4M, 2.56 mmol) at r.tfor 1 hr. Then 5-amino-2-fluoroisonicotinic acid (1.0 g, 6.41 mmol) wasadded and the resulting mixture was refluxed for 24 hours. After coolingto r.t, the solid product was collected by filteration. It was washedwith EtOAc, then dried under vacuum to afford6-fluoro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (4b, 756 mg,48.7%). 1H NMR (600 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.81 (d, J=5.6 Hz,2H), 8.14-8.06 (m, 2H), 7.74 (d, J=2.3 Hz, 1H). LCMS (m/z [M+H]⁺):243.10.

Step B: To a mixture of6-fluoro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (4b, 750 mg, 3.1mmol) in DCE (40 ml) was added thionyl chloride(1.81 ml, 24.8 mmol) andDMF (0.1 ml). The mixture was then stirred for 3 hours at 85° C. Thereaction mixture was concentrated under reduced pressure and dried undervacuum overnight. The crude product (950 mg) was used for next stepreaction without further work up or purification. LCMS (m/z [M+H]⁺):261.10.

Intermediate 5d (Scheme 5):4,5-dichloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine

Step A: A mixture of 3-bromo-5-fluoroisonicotinic acid (5a, 1.87 g, 8.5mmol) and HATU (4.85 g, 12.75 mmol) in DMF (30 ml) was added DIEA (4.5ml), the mixture was stirred at r.t for 20 minutes, thenisonicotinimidamide (1.236 g, 10.2 mmol) was added. Stirring wascontinued for another 15 hours at r.t. The reaction mixture wasconcentrated under reduced pressure. The light brown syrup crude mixturewas then dissolved in DCM and purified by silica gel chromatography(eluted with 0-10% MeOH/solvent A, solvent A is mixture of 4 liter DCMand 8 ml of 7N ammonia solution in MeOH), fractions 66-80 were pooledand concentrated to afford the desired product3-bromo-5-fluoro-N-(imino(pyridin-4-yl)methyl)isonicotinamide 5b (566mg, 20.6%). 1H NMR (500 MHz, DMSO-d6) δ 10.27 (s, 1H), 10.09 (s, 1H),8.79-8.73 (m, 2H), 8.71 (s, 2H), 7.95-7.89 (m, 2H). LCMS (m/z [M+H]⁺):323.0.

Step B: A mixture of3-bromo-5-fluoro-N-(imino(pyridin-4-yl)methyl)isonicotinamide (5b, 600mg, 1.857 mmol), DIEA (0.33 ml, 1.857 mmol), potassium carbonate (257mg, 1.857 mmol) and DBU (0.28 ml, 1.857 mmol) in DMA (8 ml) in a 20 mlmicrowave reaction vessel was heated at 150° C. for 45 minutes(microwave irradiation). The reaction mixture was diluted with water (20ml), extracted with EtOAc (3×60 ml), the desired product remained in theaqueous phase. The aqueous phase was then purified by reverse phase ISCO(10-50% CH3CN/Water) to afford the desired product5-bromo-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol 5c (520 mg, 80%purity, 74%). LCMS (m/z [M+H]⁺): 303.0.

Step C: 5-bromo-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (5c, 150 mg,0.495 mmol) was dissolved in anhydrous CH₃CN (2 ml) and followed byaddition of POCl₃ (759 mg, 4.95 mmol). The reaction mixture was thenheated at 100° C. for 16 hrs. LCMS showed the reaction was complete. Thereaction was cooled to rt and the solvent was evaporated. The residuewas diluted with ice water (40 ml), and was then extracted with EtOAc(3×40 ml). The combined organic layers were dried over Na₂SO₄, filteredand evaporated to dryness to afford the desired product4,5-dichloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine 5d (86%) Theproduct was used directly for next step without further purification. 1HNMR (400 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.87 (s, 2H), 8.68 (s, 1H), 8.16(d, J=4.9 Hz, 2H). LCMS (m/z [M+H]⁺): 277.0.

Intermediate 6b (Scheme 6, intermediate 6b′, wherein A is 4-pyridinyl):4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine

Step A: In a 20 mL microwave reactor was added PalladiumTetrakis (58.1mg, 0.050 mmol), potassium carbonate (1.256 mL, 2.51 mmol), and2,4-dichloro-1,7-naphthyridine (6a, 200 mg, 1.005 mmol) andpyridin-4-ylboronic acid (130 mg, 1.055 mmol) in acetonitrile (2 mL) togive an orange suspension. The reaction mixture was stirred at 120° C.for 60 min under microwave. The crude mixture was diluted with DCM, H₂O,separated and extracted with DCM×3. Combined the organic layers anddried Na₂SO₄, filtered and concentrated. The residue was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCMto give the product (62%). 1H NMR (400 MHz, DMSO-d6) δ 9.58 (d, J=0.9Hz, 1H), 8.85-8.78 (m, 4H), 8.32-8.29 (m, 2H), 8.11 (dd, J=5.8, 0.9 Hz,1H). LCMS (m/z [M+H]⁺): 242.1.

Synthesis of Compounds of Formula A1 Example 1:N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(Compound 1)

Title compound was prepared from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) usingstep C as in Scheme 1.

Step C: In a 20 mL vial 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine(30 mg, 0.12 mmol) was stirred in DMF (0.7 mL) at room temperature anddegassed with N₂. TEA (19 uL, 0.14 mmol) was added and stirred for 5minutes then KF (7 mg, 0.12 mmol). This mixture was stirred at roomtemperature for 15 minutes then 2-cyclopropylpropan-2-amine (0.013 mL,0.12 mmol) was added and degassed then stirred at 80° C. for two hrs.The reaction was then concentrated and purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to afford theproductN-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(50%). 1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.80 (d,J=6.1 Hz, 2H), 8.64 (d, J=5.6 Hz, 1H), 8.40 (dd, J=5.7, 0.9 Hz, 1H),8.29 (m, 2H), 7.74 (s, 1H), 1.94 (m, 1H), 1.52 (s, 6H), 0.49 (m, 4H).LCMS (m/z [M+H]⁺): 306.2.

Examples 2-110

Examples 2-110 described infra were synthesized according to theprotocol described for Example 1 using4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andvarious amines respectively except specially stated.

Example 2:N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.58(d, J=5.9 Hz, 1H), 8.31 (m, 2H), 7.89 (dd, J=5.9, 0.9 Hz, 1H), 3.90(q,J=7.0 Hz, 4H), 1.40 (t, J=7.0 Hz, 6H). LCMS (m/z [M+H]⁺): 280.1.

Example 3:N-ethyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.58(d, J=5.8 Hz, 1H), 8.30 (m, 2H), 7.88 (dd, J=5.8, 0.8 Hz, 1H), 4.95-4.90(m, 1H), 3.81(q, J=6.9 Hz, 2H), 1.40 (s, 3H), 1.38 (s, 3H), 1.35 (m,3H). LCMS (m/z [M+H]⁺): 294.2.

Example 4:2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, CDCl3) δ 9.45 (d, J=0.9 Hz, 1H), 8.83 (m, 2H), 8.78 (d,J=5.5 Hz, 1H), 8.70-8.60 (m, 2H), 7.63 (s, 1H), 5.98-5.92 (m, 1H),5.80-5.75 (m, 1H), 1.81 (d, J=7.0 Hz, 3H). LCMS (m/z [M+H]⁺): 320.1.

Example 5:N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.55(d, J=5.8 Hz, 1H), 8.31 (m, 2H), 8.03 (dd, J=5.8, 0.9 Hz, 1H), 5.15-5.10(m, 1H), 3.34 (s, 3H), 1.35 (d, J=6.6 Hz, 6H). LCMS (m/z [M+H]⁺): 280.2.

Example 6:N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.76 (m, 2H), 8.64(d, J=5.6 Hz, 1H), 8.50 (d, J=7.5 Hz, 1H), 8.32 (m, 2H), 8.28 (dd,J=5.7, 0.9 Hz, 1H), 4.74-4.67 (d, J=6.7 Hz, 1H), 1.36 (d, J=6.6 Hz, 6H).LCMS (m/z [M+H]⁺): 266.1.

Example 7:N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.80 (m, 2H), 8.64(d, J=5.8 Hz, 1H), 8.40 (dd, J=5.7, 0.9 Hz, 1H), 8.29 (m, 2H), 7.74 (s,1H), 3.85 (s, 2H), 3.28 (s, 3H), 1.60 (s, 6H). LCMS (m/z [M+H]⁺): 310.2.

Example 8:N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.9 Hz, 1H), 8.80 (m, 2H), 8.65(d, J=5.6 Hz, 1H), 8.30 (m, 2H), 8.28 (m, 1H), 7.85 (s, 1H), 3.48 (t,J=6.7 Hz, 2H), 3.20 (s, 3H), 2.35 (t, J=6.8 Hz, 2H), 1.64 (s, 6H). LCMS(m/z [M+H]⁺): 324.2.

Example 9:N-butyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.8 Hz, 1H), 8.77 (m, 2H), 8.55(d, J=5.9 Hz, 1H), 8.31 (m, 2H), 8.08 (dd, J=5.8, 0.9 Hz, 1H), 3.92 (m,2H), 3.54 (s, 3H), 1.82-1.75 (m, 2H), 1.48-1.36 (m, 2H), 0.98 (t, J=7.4Hz, 3H). LCMS (m/z [M+H]⁺): 294.2.

Example 10:N-ethyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.55(d, J=5.8 Hz, 1H), 8.31 (m, 2H), 8.05 (dd, J=5.9, 0.9 Hz, 1H), 3.94(q,J=7.1 Hz, 2H), 3.50 (s, 3H), 1.39 (t, J=7.0 Hz, 3H). LCMS (m/z [M+H]⁺):266.1.

Example 11:2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol

Step 1: In a 40 mL vial at 0° C. was added 2-amino-2-methylpropan-1-ol(1.5 g, 16.8 mmol) in 8 mL of dry DCM. DIEA (3.2 mL, 18.5 mmol) wasadded then benzyl carbonochloridate (2.37 mL, 16.8 mmol) in portions.The reaction mixture was stirred for two hrs slowly warming to roomtemperature. Solvent was evaporated under air flow. The residue waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-50% EtOAc/Hexane to afford the productbenzyl-(1-hydroxy-2-methylpropan-2-yl)carbamate (90%). LCMS (m/z[M+H]⁺): 224.3.

Step 2: In a 20 mL vial was addedbenzyl-(1-hydroxy-2-methylpropan-2-yl)carbamate (0.63 g, 2.8 mmol) in 5mL of dry THF. Potassium hydroxide (0.16 g, 2.8 mmol) was added in 0.5mL of H₂O, then tert-butyl 2-bromoacetate(0.62 mL, 4.2 mmol) andtetrabutylammonium bromide (90 mg, 0.28 mmol). The reaction mixture wasstirred overnight at 30° C. Solvent was evaporated under air flow. Theresidue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-40% EtOAc/Hexane to afford the product tert-butyl2-(2-(((benzyloxy)carbonyl)amino)-2-methylpropoxy)acetate (35%). LCMS(m/z [M+H]⁺): 338.4.

Step 3: In a 20 mL vial at 0° C. was added tert-butyl2-(2-(((benzyloxy)carbonyl)amino)-2-methylpropoxy) acetate (0.14 g, 0.17mmol) in 1 mL of dry DMF. Lithium borohydride (0.45 mL, 0.91 mmol) wasadded in portions and stirred for 4 hrs at room temperature thenquenched with water. DCM was used for extraction and the solventevaporated under air flow. The residue was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-70% EtOAc/Hexaneto afford the productbenzyl-(1-(2-hydroxyethoxy)-2-methylpropan-2-yl)carbamate (35%). LCMS(m/z [M+H]⁺): 268.3.

Step 4: In a 20 mL septa sealed vial was addedbenzyl-(1-(2-hydroxyethoxy)-2-methylpropan-2-yl)carbamate (0.03 g, 0.11mmol) in 1.2 mL of EtOH. The vial was vigorously purged with N₂ at roomtemperature. A small scoop of Pd/C (30%, cat. amount) was carefullyadded and the reaction maintained under N₂. A H₂ balloon was then usedto flush the reaction vessel thoroughly and then stirred under H₂pressure for four hrs. The H₂ was removed from the reaction then thevessel was purged with N₂. The material was then filtered through Na₂SO₄and celite and the solvent evaporated under air flow. No furtherpurification of the residue was necessary.2-(2-amino-2-methylpropoxy)ethanol (95%). 1H NMR (500 MHz, Chloroform-d)b 3.76-3.73 (m, 2H), 3.62-3.59 (m, 2H), 3.26 (s, 2H), 1.27 (s, 2H), 1.20(d, J=4.6 Hz, 1H), 1.11 (s, 6H). LCMS (m/z [M+H]⁺): 134.2.

Step 5: In a 20 mL microwave vial was added 3-aminoisonicotinamide (1a,2 g, 14.58 mmol), isonicotinaldehyde (1.521 mL, 16.04 mmol), sodiumbisulfite (1.821 g, 17.50 mmol) and 4-methylbenzenesulfonic acid hydrate(0.277 g, 1.458 mmol) in DMA (Volume: 5 mL) to give an orangesuspension. The reaction was well stirred and heated in microwave at160° C. for 12 min. The reaction mixture was diluted with water andfiltered. The solid was washed with water, MeOH and ether to give 2.03 goff white solid as the product 1b (59%). 1H NMR (400 MHz, DMSO-d6) δ9.18 (d, J=0.9 Hz, 1H), 8.88-8.78 (m, 2H), 8.73 (d, J=5.2 Hz, 1H),8.16-8.08 (m, 2H), 8.03 (dd, J=5.2, 0.9 Hz, 1H).

Step 6: In a 5 mL microwave reactor was2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (1b, 500 mg, 2.230 mmol) andphenylphosphonic dichloride (1564 microlitre, 11.15 mmol) to give abrown suspension. The reaction mixture was stirred at 170° C. for 30 minwhen LCMS indicated full conversion. The reaction mixture was quenchedwith ice/water and neutralized with saturated Na₂CO₃, then extractedwith DCM×3 and give the product 1c (74%). 1H NMR (500 MHz, DMSO-d6) δ9.65 (d, J=1.0 Hz, 1H), 8.96 (d, J=5.6 Hz, 1H), 8.87 (s, 2H), 8.47-8.30(m, 2H), 8.18 (dd, J=5.6, 1.0 Hz, 1H). LCMS (m/z [M+H]⁺): 243.1.

Step 7: In a 20 mL vial 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine(intermediate 1c) (150 mg, 0.618 mmol) was stirred in DMSO (1.5 mL) atroom temperature and degassed with N₂. DIEA (324 microlitre, 1.85 mmol)was added and stirred for 5 minutes then KF (36 mg, 0.618 mmol). Thismixture was stirred at room temperature for 15 minutes then2-(2-amino-2-methylpropoxy)ethanol (99 mg, 0.74 mmol) was added anddegassed then stirred at 60° C. for 30 min. The reaction was thenconcentrated and purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-10% MeOH/DCM to afford the title compound (25%).1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.80 (m, 2H), 8.64(d, J=5.6 Hz, 1H), 8.35 (m, 1H), 8.29 (m, 2H), 7.76 (s, 1H), 4.64-4.59(m, 1H), 3.90 (s, 2H), 3.50-3.44 (m, 4H), 1.61 (s, 6H). LCMS (m/z[M+H]⁺): 340.2

Example 12:2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol

Step 1: In a 40 ml vial at 0° C. was added 2-amino-2-methylpropan-1-ol(1.5 g, 16.8 mmol) in 8 mL of dry DCM. DIEA(3.2 mL, 18.5 mmol) was addedthen benzyl carbonochloridate (2.37 mL, 16.8 mmol) in portions. Thereaction mixture was stirred for two hrs slowly warming to roomtemperature. Solvent was evaporated under air flow. The residue waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-50% EtOAc/Hexane to afford the productbenzyl-(1-hydroxy-2-methylpropan-2-yl)carbamate (90%). 1H NMR (400 MHz,Chloroform-d) δ 7.41-7.29 (m, 5H), 5.06 (s, 2H), 4.97-4.83 (m, 1H), 4.70(s, 1H), 3.62 (s, 2H), 1.34-1.19 (m, 6H). LCMS (m/z [M+H]⁺): 224.3.

Step 2: In a 20 mL vial was addedbenzyl-(1-hydroxy-2-methylpropan-2-yl)carbamate (0.63 g, 2.8 mmol) in 5mL of dry THF. Potassium hydroxide (0.16 g, 2.8 mmol) was added in 0.5mL of H₂O, then tert-butyl 2-bromoacetate (0.62 mL, 4.2 mmol) andtetrabutylammonium bromide (90 mg, 0.28 mmol). The reaction mixture wasstirred overnight at 30° C. Solvent was evaporated under air flow. Theresidue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-40% EtOAc/Hexane to afford the product tert-butyl2-(2-(((benzyloxy)carbonyl)amino)-2-methylpropoxy)acetate (35%). 1H NMR(500 MHz, Chloroform-d) δ 7.39-7.28 (m, 5H), 5.52 (s, 1H), 5.06 (s, 2H),3.96 (s, 2H), 3.45 (s, 2H), 1.48 (s, 9H), 1.36 (s, 6H). LCMS (m/z[M+H]⁺): 338.4.

Step 3: tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)-2-methylpropoxy)acetate (700 mg, 4.15 mmol) was stirred in DCM (5 mL) in an ice bath for10 minutes. Methyl magnesium bromide (30 mL, 42 mmol) was slowly addedin portions then stirred while warming to room temp over two hrs. Thereaction was then quenched with water and extracted three times withDCM. The organic layers were combined, concentrated and purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50%EtOAc/Hexane to afford the product benzyl (1-(2-hydroxy-2methylpropoxy)-2-methylpropan-2-yl)carbamate (55%). LCMS [M+H]=296.4.

Step 4: In a 20 mL septa sealed vial was added benzyl(1-(2-hydroxy-2-methylpropoxy)-2-methylpropan-2-yl)carbamate (0.03 g,0.11 mmol) in 1.2 mL of EtOH. The vial was vigorously purged with N₂ atroom temperature. A small scoop of Pd/C (30%, cat. amount) was carefullyadded and the reaction maintained under N₂. A H₂ balloon was then usedto flush the reaction vessel thoroughly and then stirred under H₂pressure for four hrs. The H₂ was removed from the reaction then thevessel was purged with N₂. The material was then filtered through Na₂SO₄and celite and the solvent evaporated under air flow. No furtherpurification of the residue was necessary to afford(1-(2-amino-2-methylpropoxy)-2-methylpropan-2-ol) (95%). 1H NMR (500MHz, Chloroform-d) b 3.32 (s, 2H), 3.26 (s, 2H), 1.22 (s, 6H), 1.12 (s,6H). LCMS (m/z [M+H]⁺): 162.2.

Step 5: In a 20 mL microwave vial was added 3-aminoisonicotinamide (1a,2 g, 14.58 mmol), isonicotinaldehyde (1.521 mL, 16.04 mmol), sodiumbisulfite (1.821 g, 17.50 mmol) and 4-methylbenzenesulfonic acid hydrate(0.277 g, 1.458 mmol) in DMA (Volume: 5 mL) to give an orangesuspension. The reaction was well stirred and heated in microwave at160° C. for 12 min. The reaction mixture was diluted with water andfiltered. The solid was washed with water, MeOH and ether to give 2.03 goff white solid as the product 1b (59%). 1H NMR (400 MHz, DMSO-d6) δ9.18 (d, J=0.9 Hz, 1H), 8.88-8.78 (m, 2H), 8.73 (d, J=5.2 Hz, 1H),8.16-8.08 (m, 2H), 8.03 (dd, J=5.2, 0.9 Hz, 1H).

Step 6: In a 5 mL microwave reactor was2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-ol (1b, 500 mg, 2.230 mmol) andphenylphosphonic dichloride (1564 microlitre, 11.15 mmol) to give abrown suspension. The reaction mixture was stirred at 170° C. for 30 minwhen LCMS indicated full conversion. The reaction mixture was quenchedwith ice/water and neutralized with saturated Na₂CO₃, then extractedwith DCM×3 and give the product 1c (74%). 1H NMR (500 MHz, DMSO-d6) δ9.65 (d, J=1.0 Hz, 1H), 8.96 (d, J=5.6 Hz, 1H), 8.87 (s, 2H), 8.47-8.30(m, 2H), 8.18 (dd, J=5.6, 1.0 Hz, 1H). LCMS (m/z [M+H]⁺): 243.1.

Step 7: In a 20 mL vial 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine(intermediate 1c) (25 mg, 0.10 mmol) was stirred in DMSO (0.7 mL) atroom temperature and degassed with N₂. DIEA (43 uL, 0.25 mmol) was addedand stirred for 5 minutes then KF (6 mg, 0.10 mmol). This mixture wasstirred at room temperature for 15 minutes then2(1-(2-amino-2-methylpropoxy)-2-methylpropan-2-ol) (0.013 mL, 0.12 mmol)was added and degassed then stirred at 60° C. for 30 min. The reactionwas then concentrated and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to afford the titlecompound (50%). 1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.79(m, 2H), 8.64 (d, J=5.6 Hz, 1H), 8.37 (dd, 5.7, 0.9 Hz, 1H), 8.30 (m,2H), 7.80 (s, 1H), 4.40 (s, 1H), 3.90 (s, 2H), 3.17 (s, 2H), 1.61 (s,6H), 0.99 (s, 6H). LCMS (m/z [M+H]⁺): 368.2.

Example 13: N-ethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.9 Hz, 1H), 8.84 (m, 1H), 8.76(m, 2H), 8.65 (d, J=5.6 Hz, 1H), 8.34 (m, 2H), 8.18 (dd, J=5.6, 0.9 Hz,1H), 3.77-3.69(qd, J=7.2, 5.4 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H). LCMS (m/z[M+H]⁺): 252.1.

Example 14: N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.82 (t, J=5.5 Hz,1H), 8.78 (m, 2H), 8.64 (d, J=5.5 Hz, 1H), 8.33 (m, 2H), 8.19 (dd,J=5.6, 0.9 Hz, 1H), 3.70-3.62(td, J=7.0, 5.7 Hz, 2H), 1.80-1.70 (m, 2H),1.00 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 266.1.

Example 15:N-(2-cyclohexylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.16 (d, J=0.9 Hz, 1H), 8.80 (m, 2H), 8.62(d, J=5.6 Hz, 1H), 8.40 (dd, J=5.7, 0.9 Hz, 1H), 8.28 (m, 2H), 7.62 (s,1H), 1.76-1.70 (m, 4H), 1.62-1.59 (m, 1H), 1.52 (s, 6H), 1.18-1.04(q,J=11.8, 10.9 Hz, 6H). LCMS (m/z [M+H]⁺): 348.2.

Example 16:N-(3-methyloxetan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.9 Hz, 1H), 9.14 (s, 1H), 8.76(m, 2H), 8.68 (d, J=5.5 Hz, 1H), 8.25 (m, 2H), 8.16 (dd, J=5.6 Hz, 1H),4.90 (d, J=6.3 Hz, 2H), 4.64 (d, J=6.5 Hz, 2H), 1.82 (s, 3H)). LCMS (m/z[M+H]⁺): 294.1.

Example 17:N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=1.0 Hz, 1H), 8.78 (m, 2H),8.66(m, 1H), 8.33 (m, 2H), 8.32 (m, 1H), 7.91 (m, 1H), 4.45-4.42 (m,1H), 2.20-2.14 (m, 1H), 1.98-1.82 (m, 2H), 1.80-1.72 (m, 2H), 1.70-1.62(m, 1H), 1.50-1.42 (m, 1H), 0.91 (d, J=7.0 Hz, 3H). LCMS (m/z [M+H]⁺):306.2.

Example 17b:N-((1R,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(17a) andN-((1S,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(17b)

To a solution of 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (130mg, 0.536 mmol) in DMF (10 ml) was added TEA (0.23 ml, 1.61 mmol) and KF(32.7 mg, 0.56 mmol). The mixture was stirred for 5 minute beforecis-2-methylcyclopentanamine hydrochloride (72.7 mg, 0.536 mmol) wasadded. The resulting mixture was then stirred for 2 hours at 50° C. Thecrude mixture was then purified by silica gel chromatography to afford109 mg product. 100 mg of this material was subjected to chiralseparation to afford two cis isomers, peak 1 (T_(R)=1.46 min) isomer 35mg, peak 2 (T_(R)=1.95 min) isomer 46 mg. Chiral center assignments aretentative chiral separation conditions: solvent A CO₂ (80%), solvent BMeOH+0.1% NH₄Cl (20%), flow rate 2 ml/min, column 21×250 mm AD-H, runtime 6 minute stacked injections, 10 minute elution time.

Peak 1 (T_(R)=1.46 min) isomer: 1H NMR (500 MHz, DMSO-d6) δ 9.19 (s,1H), 8.77 (d, J=5.8 Hz, 2H), 8.66 (d, J=5.6 Hz, 1H), 8.40 (dd, J=5.7,0.8 Hz, 1H), 8.36-8.29 (m, 3H), 4.86 (p, J=7.5 Hz, 1H), 2.09 (dtd,J=11.7, 8.1, 3.4 Hz, 1H), 1.89 (dddq, J=29.7, 12.7, 8.4, 3.8 Hz, 3H),1.66-1.53 (m, 1H), 1.51-1.41 (m, 1H), 0.83 (d, J=7.1 Hz, 3H). LCMS (m/z[M+H]⁺): 306.2.

Peak 2 (T_(R)=1.95 min) isomer: 1H NMR (500 MHz, DMSO-d6) δ 9.19 (d,J=0.7 Hz, 1H), 8.80-8.74 (m, 2H), 8.66 (d, J=5.6 Hz, 1H), 8.40 (dd,J=5.7, 0.8 Hz, 1H), 8.36-8.29 (m, 3H), 4.86 (p, J=7.6 Hz, 1H), 2.09 (dp,J=12.4, 4.6, 4.2 Hz, 1H), 1.98-1.79 (m, 3H), 1.66-1.54 (m, 1H),1.50-1.41 (m, 1H), 0.83 (d, J=7.1 Hz, 3H). LCMS (m/z [M+H]⁺): 306.2.

Example 18:3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol

1H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J=5.4 Hz, 1H), 8.79 (m, 2H), 8.63(m, 1H), 8.33 (m, 1H), 8.28 (dd, J=5.4, 0.8 Hz, 2H), 7.58 (s, 1H), 5.00(d, J=5.6 Hz, 1H), 4.30-4.26 (m, 1H), 1.29 (s, 3H), 1.27 (s, 3H), 1.05(d, J=6.7 Hz, 3H). LCMS (m/z [M+H]⁺): 310.2.

Example 19: N-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.9 Hz, 1H), 8.82(m, 1, 2H), 8.65(d, J=5.6 Hz, 1H), 8.34 (m, 2H), 8.20 (dd, J=5.6, 0.9 Hz, 1H),3.75-3.68(td, 7.2, 5.6 Hz, 2H), 1.78-1.69 (m, 2H), 1.50-1.40 (m, 2H),0.98 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 280.2.

Example 20:N-(2-methyl-4-phenylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.63(d, J=5.6 Hz, 1H), 8.40 (dd, J=5.7, 0.9 Hz, 1H), 8.30 (m, 2H), 7.79 (s,1H), 7.13-7.04 (m, 5H), 2.61-2.56 (dd, J=10.7, 6.0 Hz, 2H), 2.48-2.43(m, 2H), 1.65 (s, 6H). LCMS (m/z [M+H]⁺): 370.2.

Example 21: N-cyclopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.9 Hz, 1H), 8.83 (m, 1H), 8.78(m, 2H), 8.64 (d, J=5.6 Hz, 1H), 8.40 (m, 2H), 8.18(dd, J=5.6 1.0 Hz,1H), 3.30-3.22 (m, 1H), 0.98-0.94 (m, 2H), 0.80-0.76 (m, 2H). LCMS (m/z[M+H]⁺): 264.1.

Example 22:N-(4-methanesulfonyl-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.79 (m, 2H), 8.65(d, J=5.6 Hz, 1H), 8.39 (dd, J=5.7, 0.9 Hz, 1H), 8.30 (m, 2H), 7.76 (s,1H), 3.15-3.10 (m, 2H), 2.90 (s, 3H), 2.65-2.60 (m, 2H), 1.61 (s, 6H).LCMS (m/z [M+H]⁺): 372.1.

Example 23:2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol

1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.79 (m, 2H), 8.63(d, J=5.6 Hz, 1H), 8.32 (dd, J=5.7, 0.9 Hz, 1H), 8.29 (m, 2H), 7.38 (s,1H), 4.81 (t, J=6.0 Hz, 2H), 3.97-3.94 (dd, J=10.8, 6.0 Hz, 2H),3.90-3.82 (dd, J=10.9, 6.2 Hz, 2H), 1.52 (s, 3H). LCMS (m/z [M+H]⁺):312.1.

Example 24:3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol

1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.77 (m, 2H), 8.64(d, J=5.6 Hz, 1H), 8.38 (m, 1H), 8.32 (m, 2H), 8.30 (s, 1H), 4.80 (d,J=5.5 Hz, 1H), 4.76-4.72 (m, 1H), 3.93-3.88 (m, 1H), 1.31 (d, J=6.7 Hz,3H), 1.19 (d, J=6.3 Hz, 3H). LCMS (m/z [M+H]⁺): 296.2.

Example 25:2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)aceticacid

Title compound was prepared from tert-butyl2-(2-amino-2-methylpropoxy)acetate and4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine as described in Step C,Example 1, followed by de-protection of the tert-butyl ester.

De-protection: tert-butyl2-(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propoxy)yl)amino)propoxy)acetate (25 mg, 0.061 mmol) was stirred in a40% mixture of TFA in DCM at room temperature for two hours. No startingmaterial was observed by TLC or LCMS. The reaction was diluted with DCMand concentrated using N₂ and mild heat then repeated three times andput on high vacuum overnight. Intermediates under these conditions weretypically used without further purification. Title compounds under theseconditions were then also purified by flash chromatography. 1H NMR (400MHz, DMSO-d6) δ 9.20(d, 0.8 Hz, 1H), 8.80 (m, 2H), 8.63 (d, J=5.6 Hz,1H), 8.33(ddd, J=10.8, 5.1, 1.2 Hz, 2H), 8.30 (m, 1H), 8.19 (s, 1H),4.14 (s, 2H), 3.80 (s, 2H), 3.16 (s, 1H), 1.62 (s, 6H). LCMS (m/z[M+H]⁺): 354.2.

Example 26:(1R,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.78 (m, 1H), 8.65(m, 1H), 8.40 (m, 1H), 8.37 (m, 1H), 8.32 (m, 2H), 4.72 (d, J=3.7 Hz,1H), 4.60-4.52(ddd, J=15.9, 9.2, 4.4 Hz, 1H), 4.44-4.40 (m, 1H),2.04-1.98 (m, 3H), 1.90-1.82 (m, 1H), 1.74-1.56 (m, 2H). LCMS (m/z[M+H]⁺): 308.1.

Example 27:4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.30 (d, J=0.8 Hz, 1H), 8.85 (m, 1H), 8.80(m, 2H), 8.74 (m, 1H), 8.39 (d, J=4.3 Hz, 1H), 8.33 (dd, J=5.7, 0.9 Hz,2H), 5.76-5.70 (m, 1H), 4.77-4.70 (m, 1H), 3.61-3.55 (m, 2H), 2.11-2.08(m, 2H). LCMS (m/z [M+H]⁺): 350.1.

Example 28:N-(1-methanesulfonyl-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.9 Hz, 1H), 8.79 (m, 2H), 8.67(dd, J=5.9, 4.7 Hz, 1H), 8.40 (dd, J=5.7, 0.9 Hz, 1H), 8.30 (m, 2H),8.08 (s, 1H), 4.13 (s, 2H), 2.90 (s, 3H), 1.80 (s, 6H). LCMS (m/z[M+H]⁺): 358.1.

Example 29:(2S)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid

1H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J=0.8 Hz, 1H), 8.79 (m, 2H), 8.76(m, 1H), 8.70 (m, 1H), 8.30 (dd, J=5.6, 0.8 Hz, 2H), 7.81 (s, 1H), 2.02(s, 3H). LCMS (m/z [M+H]⁺): 364.1.

Example 30:2-[(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)amino]aceticacid

Title compound was prepared using tert-butyl2-((2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)amino)acetateas described in the above scheme. 1H NMR (400 MHz, DMSO-d6) δ 9.25 (m,1H), 8.82 (m, 2H), 8.70 (m, 1H), 8.40 (m, 1H), 8.35 (m, 2H), 7.84 (d,J=9.4 Hz, 1H), 3.89-3.80 (d, J=6.2 Hz, 1H), 3.60-3.56 (d, J=11.0 Hz,4H), 1.70 (s, 6H). LCMS (m/z [M+H]⁺): 353.2.

Step 1:2-methyl-N2-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine(20 mg, 0.068 mmol) was stirred in DCM/DMF at room temperature. 21microlitre of TEA (21 microlitre, 0.149 mmol) was added and stirred forthree minutes. tert-butyl 2-bromoacetate (11 microlitre, 0.071 mmol) anda catalytic amount of DMAP was then added and stirred at roomtemperature for four hrs. Reaction was then concentrated and purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCMto afford the product tert-butyl2-((2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)amino)acetate(35%). LCMS (m/z [M+H]⁺): 409.5.

Example 31:(2R)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid

1H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J=0.8 Hz, 1H), 8.79 (m, 2H), 8.77(m, 1H), 8.70 (m, 1H), 8.30 (m, 2H), 7.83 (s, 1H), 2.02 (s, 3H). LCMS(m/z [M+H]⁺): 364.1.

Example 32: Methyl2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoate

1H NMR (400 MHz, DMSO-d6) δ 9.24 (d, J=0.8 Hz, 1H), 8.85 (s, 1H), 8.79(m, 2H), 8.70 (d, J=5.5 Hz, 1H), 8.39 (dd, J=5.7, 1.0 Hz, 1H), 8.25 (m,2H), 3.51 (s, 3H), 1.69 (s, 6H). LCMS (m/z [M+H]⁺): 324.1.

Example 33:(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.65(d, J=5.6 Hz, 1H), 8.51 (d, J=7.0 Hz, 1H), 8.38 (m, 1H), 8.36 (m, 1H),8.31(dd, J=5.6, 0.9, Hz, 1H), 4.92 (d, J=4.6 Hz, 1H), 4.60-4.54 (d,J=7.0 Hz, 1H), 4.26-4.20 (m, 1H), 2.32-2.22(ddt, J=13.2, 8.2, 4.3 Hz,1H), 2.00-1.92 (m, 1H), 1.85-1.72 (m, 2H), 1.70-1.56 (m, 2H). LCMS (m/z[M+H]⁺): 308.1.

Example 34:2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid

Title compound was prepared from de-protection of methyl2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoateas described in Step A, Example 25.

1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 9.20 (d, J=0.8 Hz, 1H),8.86-8.80 (s, 1H), 8.75 (m, 2H), 8.67 (d, J=5.6 Hz, 1H), 8.30 (m, 2H),8.28 (m, 1H), 1.66 (s, 6H). LCMS (m/z [M+H]⁺): 310.1.

Example 35:2-(2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethoxy)ethan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.9 Hz, 1H), 8.89 (t, J=5.5 Hz,1H), 8.76 (m, 2H), 8.65 (d, J=5.5 Hz, 1H), 8.32 (m, 2H), 8.20 (dd,J=5.7, 0.9 Hz, 1H), 4.60-4.57 (m, 1H), 3.89(q, J=5.7 Hz, 2H), 3.78 (t,J=5.8 Hz, 2H), 3.51-3.48 (d, J=2.9 Hz, 4H). LCMS (m/z [M+H]⁺): 312.1.

Example 36:2-(hydroxymethyl)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.80 (m, 2H), 8.65(d, J=5.6 Hz, 1H), 8.30(ddd, J=19.4, 5.1, 1.3 Hz, 1H), 8.26 (m, 2H),7.19 (s, 1H), 4.72 (t, J=6.0 Hz, 3H), 4.00 (d, J=6.0 Hz, 6H). LCMS (m/z[M+H]⁺): 328.1.

Example 37:3-methyl-3-(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanamido)butanoic acid

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.9 Hz, 1H), 8.80(dt, J=4.5, 1.1Hz, 2H), 8.64 (m, 1H), 8.33 (m, 2H), 8.30 (m, 1H), 7.90 (s, 1H), 1.72(s, 6H), 1.70 (s, 2H), 1.30 (s, 2H), 1.00 (s, 6H). LCMS (m/z [M+H]⁺):423.2.

Example 38:2-(pyridin-4-yl)-N-(1,1,1-trifluoro-3-phenylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.9 Hz, 1H), 8.99 (d, J=9.0 Hz,1H), 8.75 (m, 2H), 8.71 (d, J=5.6 Hz, 1H), 8.33 (m, 1H), 8.30 (m, 2H),7.41 (m, 2H), 7.11 (m, 2H), 7.02 (m, 1H), 5.95-5.86 (t, J=8.2 Hz, 1H),3.38-3.34 (m, 1H), 3.24-3.17(ddt, J=13.8, 11.7, 0.7 Hz, 1H). LCMS (m/z[M+H]⁺): 396.1.

Example 39:N-{[4-(dimethylamino)oxan-4-yl]methyl}-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.78 (m, 2H), 8.67 (d, J=5.5Hz, 1H), 8.52 (s, 1H), 8.33 (m, 2H), 8.28 (m, 1H), 3.62 (t, J=10.6 Hz,2H), 3.53 (d, J=10.7 Hz, 2H), 3.27 (d, J=5.3 Hz, 2H), 2.41 (s, 6H), 1.80(d, J=14.1 Hz, 2H), 1.63 (t, J=11.5 Hz, 2H). LCMS (m/z [M+H]⁺): 365.2.

Example 40:3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid

1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 9.20 (d, J=0.8 Hz, 1H), 8.80(m, 2H), 8.66 (m, 1H), 8.40 (m, 1H), 8.30 (m, 2H), 7.99 (m, 1H),3.12-3.08(q, J=7.3 Hz, 2H), 1.70(s, 6H. LCMS (m/z [M+H]⁺): 324.1.

Example 41:N-(2-methanesulfonylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 9.10(t, J=5.6 Hz, 1H), 8.78(m, 2H), 8.69 (d, J=5.7 Hz, 1H), 8.39 (m, 2H), 8.14 (d, J=5.7 Hz, 1H),4.13-4.09(q, J=6.4 Hz, 2H), 3.62 (t, J=6.7 Hz, 2H), 3.09 (s, 3H). LCMS(m/z [M+H]⁺): 330.1.

Example 42:N-[2-(adamantan-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.16 (d, J=0.8 Hz, 1H), 8.78 (m, 2H), 8.63(d, J=5.6 Hz, 1H), 8.45 (m, 1H), 8.25 (m, 2H), 7.06 (s, 1H), 1.99-1.96(d, J=7.5 Hz, 3H), 1.80-1.76 (m, 6H), 1.67-1.64 (m, 6H), 1.64-1.60 (m,6H). LCMS (m/z [M+H]⁺): 400.2.

Example 43:2-methyl-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propanamide

1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.49 (m, 1H), 8.84 (m, 2H),8.80 (m, 1H), 8.40 (m, 2H), 8.25 (dd, J=5.8, 1.0 Hz, 1H), 3.29-3.18 (m,1H), 1.26 (d, J=6.8 Hz, 6H). LCMS (m/z [M+H]⁺): 294.1.

Example 44:4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid

1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J=0.9 Hz, 1H), 8.80 (m, 2H), 8.71(t, J=4.9 Hz, 1H), 8.34 (m, 2H), 8.23 (d, J=5.7 Hz, 1H), 5.84-5.79 (m,1H), 3.65-3.55 (d, J=4.8 Hz, 1H), 2.45-2.41 (d, J=4.7 Hz, 1H), 2.32-2.26(m, 1H). LCMS (m/z [M+H]⁺): 364.1.

Example 45:N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propane-2-sulfonamide

1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.88 (m, 2H), 8.76 (d, J=5.6Hz, 1H), 8.40-8.30 (m, 3H), 4.20-4.10(q, J=7.1 Hz, 1H), 1.40 (d, J=6.9Hz, 6H). LCMS (m/z [M+H]⁺): 330.1.

Example 46:2-(pyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (m, 1H), 9.18 (s, 1H), 8.75 (m, 2H),8.64 (d, J=5.6 Hz, 1H), 8.30 (m, 2H), 8.21 (dd, J=5.6, 1.0 Hz, 1H),3.81-3.75(td, J=6.9, 5.3 Hz, 2H), 2.95 (t, J=7.2 Hz, 2H), 2.18-2.10 (m,2H). LCMS (m/z [M+H]1): 334.2.

Example 47:N-methyl-2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

Step 1: A mixture of urea (40.00 g, 666.00 mmol) and 3-aminoisonicotinicacid (2a, 18.40 g, 133.20 mmol) was heated at 210° C. for 1 hr (NOTE: nosolvent was used). NaOH (2N, 320 mL) was added, and the mixture wasstirred at 90° C. for 1 h. The solid was collected by filtration, andwashed with water. The crude product thus obtained was suspended in HOAc(400 mL), and stirred at 100° C. for 1 h. The mixture was cooled to RT,filtered, and the solid was washed with a large amount of water, andthen dried under the vacuum to givepyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b, 17.00 g, 78% yield)without further purification. LCMS (m/z [M+H]⁺): 164.0.

Step 2: To a mixture of pyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b,20.00 g, 122.60 mmol) and POCl₃ (328.03 g, 2.14 mol) in toluene (200 mL)was added DIEA (31.69 g, 245.20 mmol) dropwise and this reaction mixturestirred at 25° C. overnight (18 hr) to give suspension.

The solvent and POCl₃ was removed under vacuum, diluted with DCM (50mL), neutralized with DIEA to pH=7 at −20° C. and concentrated again,the residue was purified by column (20-50% EA/PE) to give2,4-dichloropyrido[3,4-d]pyrimidine (2c, 20.00 g, 99.99 mmol, 82% yield)as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 9.52 (s, 1H), 8.92(d, J=5.6 Hz, 1H), 8.04 (d, J=5.6 Hz, 1H). LCMS (m/z [M+H]⁺): 200.0.

Step 3: In a 20 mL vial 2,4-dichloropyrido[3,4-d]pyrimidine (600 mg, 3.0mmol) was stirred in DMSO (0.7 mL) at room temperature and degassed withN₂. DIEA (1 mL, 6 mmol) was added and stirred for 5 minutes then KF (174mg, 3 mmol). This mixture was stirred at room temperature for 15 minutesthen racemic 1,1,1-trifluoro-N-methylpropan-2-amine (419 mg, 3.3 mmol)was added and degassed then stirred at 60° C. for 4 hours. The reactionwas then concentrated and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to afford2-chloro-N-methyl-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine(680 mg, 74%). 1H NMR (500 MHz, Acetone-d6) δ 9.09 (d, J=0.9 Hz, 1H),8.59 (d, J=5.9 Hz, 1H), 8.22 (dd, J=5.9, 0.9 Hz, 1H), 5.93 (dddd,J=15.3, 8.3, 7.0, 1.2 Hz, 1H), 3.61 (q, J=1.0 Hz, 3H), 1.63 (d, J=7.0Hz, 3H). LCMS (m/z [M+H]⁺): 291.7.

Step 4: In a 20 mL microwave reactor was added PalladiumTetrakis (99 mg,0.086 mmol), potassium carbonate (2.15 mL, 4.3 mmol), and 2chloro-N-methyl-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine(500 mg, 1.72 mmol) and pyridin-4-ylboronic acid (233 mg, 1.89 mmol) inacetonitrile (8 mL) to give an yellow suspension. The reaction mixturewas stirred at 130° C. for 30 min under microwave. The crude mixture wasdiluted with DCM, H₂O, separated and extracted with DCM×3. Combined theorganic layers and dried Na₂SO₄, filtered and concentrated. The residuewas purified by flash chromatography on a COMBIFLASH® system (ISCO)using 0-10% MeOH/DCM to give Example 47, the racemic product, thenfollowed by chiral HPLC (21×250 mm OJ-H column with 85% CO₂ as phase Aand 15% MeOH as phase B, flow rate 2 mL/min, 30° C., 3.5 min elutiontime) to separate the enantiomers to afford Examples 4ba and 48b.

Example 48a:N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.33 (d, J=0.8 Hz, 1H), 8.86-8.75 (m, 2H),8.63 (d, J=5.9 Hz, 1H), 8.38-8.30 (m, 2H), 8.20 (dd, J=6.0, 0.9 Hz, 1H),6.11 (qt, J=8.5, 7.4 Hz, 1H), 3.50 (d, J=1.1 Hz, 3H), 1.61 (d, J=7.0 Hz,3H). LCMS (m/z [M+H]⁺): 334.1. Chiral HPLC T_(R)=1.73 min. Absolutestereochemistry was confirmed by X-ray crystal structure.

Example 48b:N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.33 (d, J=0.8 Hz, 1H), 8.86-8.75 (m, 2H),8.63 (d, J=5.9 Hz, 1H), 8.38-8.30 (m, 2H), 8.20 (dd, J=6.0, 0.9 Hz, 1H),6.11 (qt, J=8.5, 7.4 Hz, 1H), 3.50 (d, J=1.1 Hz, 3H), 1.61 (d, J=7.0 Hz,3H). LCMS (m/z [M+H]⁺): 334.1. Chiral HPLC T_(R)=1.25 min. Absolutestereochemistry was confirmed by X-ray crystal structure.

Example 49:2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol

1H NMR (400 MHz, Acetone-d6) δ 9.57 (s, 1H), 9.15 (d, J=0.9 Hz, 1H),8.82-8.72 (m, 2H), 8.56 (d, J=5.6 Hz, 1H), 8.44-8.37 (m, 2H), 7.69 (dd,J=5.6, 0.9 Hz, 1H), 2.08 (s, 2H), 1.87 (s, 6H), 1.48 (d, J=0.8 Hz, 6H).LCMS (m/z [M+H]⁺): 338.2.

Example 50:4,4,4-trifluoro-2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol

The title compound was prepared from methyl2-amino-3,3,3-trifluoro-2-methylpropanoate hydrochloride and4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine as described in Step C,Example 1, followed by Grignard reaction with methylmagnesium bromide.

Grignard reaction: methyl3,3,3-trifluoro-2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate(7 mg, 0.019 mmol) was stirred with methylmagnesium bromide (1.4 M inhexanes, 0.133 mL) in DCM at 0° C. for 1 hour. No starting material wasobserved by TLC or LCMS. The reaction was quenched by MeOH andconcentrated. The residue was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to give the titlecompound (66%). 1H NMR (500 MHz, Methanol-d4) δ 9.28 (d, J=0.9 Hz, 1H),8.77-8.71 (m, 2H), 8.67 (d, J=5.7 Hz, 1H), 8.44-8.38 (m, 2H), 7.85 (dd,J=5.7, 0.9 Hz, 1H), 2.09 (d, J=1.4 Hz, 3H), 1.55 (t, J=2.2 Hz, 3H), 1.37(s, 3H). LCMS (m/z [M+H]⁺): 378.2.

Example 51:(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentyl)methanol

1H NMR (500 MHz, Chloroform-d) δ 9.35 (d, J=0.9 Hz, 1H), 8.82-8.75 (m,2H), 8.65 (d, J=5.6 Hz, 1H), 8.30-8.24 (m, 2H), 7.49 (dd, J=5.8, 0.9 Hz,1H), 3.99 (d, J=4.0 Hz, 2H), 2.19 (td, J=7.3, 6.7, 2.5 Hz, 4H),1.94-1.78 (m, 4H). LCMS (m/z [M+H]⁺): 322.2.

Example 52:N-(3-methoxycyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.19 (d, J=0.9 Hz, 1H), 8.87 (d, J=6.4 Hz,1H), 8.80-8.72 (m, 2H), 8.66 (d, J=5.5 Hz, 1H), 8.37-8.32 (m, 2H), 8.25(dd, J=5.6, 0.9 Hz, 1H), 4.53-4.42 (m, 1H), 3.84-3.74 (m, 1H), 3.20 (s,3H), 2.89-2.79 (m, 2H), 2.11 (tdd, J=9.0, 7.5, 2.8 Hz, 2H). LCMS (m/z[M+H]⁺): 308.1.

Example 53:(1R,2R)-1-N,2-N-dimethyl-1-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]cyclohexane-1,2-diamine

1H NMR (500 MHz, Methanol-d4) δ 9.23 (d, J=0.8 Hz, ¹H), 8.73-8.68 (m,2H), 8.53 (d, J=5.9 Hz, 1H), 8.46-8.41 (m, 2H), 8.22 (dd, J=5.8, 0.9 Hz,1H), 3.47 (s, 3H), 2.91 (dd, J=13.0, 9.0 Hz, 1H), 2.39 (s, 3H), 2.30(dtt, J=12.6, 5.1, 2.5 Hz, 1H), 2.04 (dp, J=12.4, 3.1 Hz, 1H), 1.90(dddd, J=23.1, 13.0, 5.6, 3.0 Hz, 2H), 1.84-1.74 (m, 1H), 1.58 (qt,J=12.7, 3.5 Hz, 1H), 1.42 (qt, J=13.2, 3.3 Hz, 1H), 1.33-1.23 (m, 1H).LCMS (m/z [M+H]⁺): 349.2.

Example 54: methyl(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate

1H NMR (500 MHz, DMSO-d6) δ 9.20 (d, J=0.9 Hz, 1H), 8.95 (d, J=6.0 Hz,1H), 8.82-8.72 (m, 2H), 8.67 (d, J=5.5 Hz, 1H), 8.34-8.28 (m, 2H), 8.25(dd, J=5.6, 1.0 Hz, 1H), 4.95 (h, J=7.3 Hz, 1H), 3.71 (s, 3H), 3.27-3.21(m, 1H), 2.72 (dddd, J=10.7, 8.2, 4.3, 2.5 Hz, 2H), 2.62-2.51 (m, 2H).LCMS (m/z [M+H]⁺): 336.1.

Example 55: ethyl1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate

1H NMR (400 MHz, Chloroform-d) δ 9.27 (dd, J=12.0, 4.4 Hz, 1H),8.83-8.74 (m, 2H), 8.54-8.40 (m, 1H), 8.40-8.31 (m, 2H), 7.56-7.49 (m,1H), 4.32-4.16 (m, 2H), 3.03-2.85 (m, 2H), 2.49 (dddd, J=11.8, 9.4, 7.1,2.3 Hz, 2H), 2.31-2.11 (m, 2H), 1.22 (dtd, J=9.6, 7.5, 7.0, 1.8 Hz, 3H).LCMS (m/z [M+H]⁺): 350.2.

Example 56:1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid

The title compound was prepared by hydrolysis of the ethyl ester ofExample 55.

Hydrolysis: a suspension of example 55 (27 mg, 0.077 mmol) and lithiumhydroxide (0.077 mL, 0.077 mmol) in MeOH (2 mL) was stirred at 90° C.for 3 hr until LCMS indicated no starting material left. The reactionmixture was neutralized with 2 mL 1M HCl. The aqueous layer was backextracted with DCM×5. The combined organic layer was washed with brine,dried and concentrated. The residue was washed with DCM/ether to givethe title compound (92%). 1H NMR (500 MHz, DMSO-d6) δ 12.49 (s, 1H),9.35 (s, 1H), 9.23 (d, J=0.9 Hz, 1H), 8.80-8.72 (m, 2H), 8.70 (d, J=5.5Hz, 1H), 8.34 (dd, J=5.6, 1.0 Hz, 1H), 8.32-8.21 (m, 2H), 2.82 (ddd,J=13.3, 8.8, 4.9 Hz, 2H), 2.49-2.43 (m, 2H), 2.12-1.96 (m, 2H). LCMS(m/z [M+H]⁺): 322.1.

Example 57:(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid

The title compound was prepared by hydrolysis of the ethyl ester ofExample 54 using the procedure as described in Example 56. 1H NMR (500MHz, DMSO-d6) δ 9.26 (d, J=0.9 Hz, 1H), 9.21 (d, J=6.0 Hz, 1H),9.02-8.93 (m, 2H), 8.73 (d, J=5.6 Hz, 1H), 8.66 (d, J=5.7 Hz, 2H), 8.38(dd, J=5.7, 0.9 Hz, 1H), 4.92 (q, J=7.4 Hz, 1H), 3.19-3.11 (m, 1H),2.76-2.68 (m, 2H), 2.56 (ddd, J=12.9, 6.5, 2.7 Hz, 2H). LCMS (m/z[M+H]⁺): 322.1.

Example 58:2-(pyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.27 (d, J=0.8 Hz, 1H), 8.84-8.77 (m, 2H),8.72 (d, J=5.7 Hz, 1H), 8.51 (dd, J=5.7, 0.9 Hz, 1H), 8.30-8.24 (m, 2H),7.98 (s, 1H), 1.91 (s, 6H). LCMS (m/z [M+H]⁺): 334.1.

Example 59: N-tert-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.82-8.77 (m, 2H),8.64 (d, J=5.6 Hz, 1H), 8.39 (dd, J=5.7, 0.9 Hz, 1H), 8.34-8.29 (m, 2H),7.88 (s, 1H), 1.66 (s, 9H). LCMS (m/z [M+H]⁺): 280.2.

Example 60:N-(1-methylcyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J=0.8 Hz, 1H), 8.77 (dd, J=6.3, 1.9Hz, 2H), 8.63 (d, J=5.6 Hz, 1H), 8.33-8.29 (m, 2H), 8.27 (dd, J=5.7, 0.9Hz, 1H), 2.70-2.64 (m, 1H), 2.58-2.53 (m, 1H), 2.35-2.26 (m, 2H),2.04-1.81 (m, 2H), 1.71 (s, 3H). LCMS (m/z [M+H]⁺): 292.2.

Example 61:3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol

1H NMR (500 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.83-8.77 (m, 2H),8.65 (d, J=5.6 Hz, 1H), 8.34-8.29 (m, 2H), 8.17 (s, 1H), 8.12 (dd,J=5.7, 0.9 Hz, 1H), 4.86 (s, 1H), 3.68-3.61 (m, 2H), 2.19 (t, J=6.6 Hz,2H), 1.66 (s, 6H). LCMS (m/z [M+H]⁺): 310.2.

Example 62:2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.26 (d, J=0.9 Hz, 1H), 8.98 (s, 1H),8.81-8.76 (m, 2H), 8.71 (d, J=5.6 Hz, 1H), 8.39 (dd, J=5.8, 0.9 Hz, 1H),8.30-8.25 (m, 2H), 2.91-2.76 (m, 4H), 2.11-1.96 (m, 2H). LCMS (m/z[M+H]⁺): 346.1.

Example 63:N-(2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.19-9.12 (m, 1H), 8.72 (ddt, J=6.3,4.7, 1.6 Hz, 2H), 8.57 (dd, J=5.8, 2.6 Hz, 1H), 8.43 (tt, J=7.3, 3.4 Hz,2H), 8.24-8.17 (m, 1H), 2.23 (qd, J=6.7, 6.0, 2.6 Hz, 2H), 1.66 (s, 6H),0.94 (td, J=7.4, 1.6 Hz, 3H). LCMS (m/z [M+H]⁺): 294.2.

Example 64:2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.40 (s, 1H), 9.29 (d, J=0.9 Hz, 1H),8.83-8.78 (m, 2H), 8.71 (d, J=5.6 Hz, 1H), 8.40-8.35 (m, 2H), 8.27 (dd,J=5.7, 1.0 Hz, 1H), 1.65-1.55 (m, 2H), 1.41-1.37 (m, 2H). LCMS (m/z[M+H]⁺): 332.1.

Example 65: N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

In a 20 mL vial 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine(intermediate 1c) (200 mg, 0.82 mmol) was stirred in DCM (5 mL) at roomtemperature and degassed with N₂. DIEA (324 microlitre, 1.85 mmol) wasadded and stirred for 5 minutes then KF (48 mg, 0.82 mmol). This mixturewas stirred at room temperature for 15 minutes then cyclopentanamine (84mg, 0.99 mmol) was added and degassed then stirred at 25° C. for 16hours. The reaction was then concentrated and purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCM toafford the title compound (51%). 1H NMR (400 MHz, DMSO-d6) 9.20 (s, 1H),8.78 (d, 2H), 8.55 (d, 1H), 8.31 (d, 2H), 8.03 (d, 1H), 5.15-5.10 (m,1H), 3.34 (s, 3H), 1.35 (s, 6H). LCMS (m/z [M+H]⁺): 292.2.

Example 66:2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.82-8.77 (m, 2H),8.64 (d, J=5.6 Hz, 1H), 8.38 (dd, J=5.7, 0.9 Hz, 1H), 8.30 (dt, J=4.5,1.7 Hz, 2H), 7.62 (s, 1H), 4.95-4.89 (m, 1H), 3.86 (d, J=6.1 Hz, 2H),1.57 (s, 5H). LCMS (m/z [M+H]⁺): 296.1.

Example 67:3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol

The title compound was prepared from methyl2-amino-3,3,3-trifluoro-2-methylpropanoate hydrochloride and4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine as described in Step C,Example 1, followed by reduction of methyl ester to alcohol with lithiumaluminum hydride.

Reduction: methyl3,3,3-trifluoro-2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate(12 mg, 0.032 mmol) was stirred with lithium aluminum hydride (4.8 mg,0.127 mmol) in THF at 0° C. for 25 hours. No starting material wasobserved by TLC or LCMS. The reaction mixture was diluted with DCM/MeOH,washed by H₂O and brine, then concentrated. The residue was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCMto give the title compound (17%). 1H NMR (500 MHz, DMSO-d6) δ 8.37 (d,J=1.0 Hz, 1H), 7.86-7.81 (m, 2H), 7.74 (d, J=5.7 Hz, 1H), 7.54-7.49 (m,2H), 7.32 (dt, J=5.7, 1.3 Hz, 1H), 3.77 (d, J=11.6 Hz, 1H), 3.19-3.12(m, 1H), 1.02 (d, J=1.2 Hz, 3H). LCMS (m/z [M+H]⁺): 350.1.

Example 68: N-(butan-2-yl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.9 Hz, 1H), 8.80-8.74 (m, 2H),8.65 (d, J=5.5 Hz, 1H), 8.43 (d, J=7.8 Hz, 1H), 8.36-8.27 (m, 3H), 4.56(hept, J=6.4 Hz, 1H), 1.83-1.63 (m, 2H), 1.33 (d, J=6.6 Hz, 3H), 0.97(t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 280.2.

Example 68a:(S)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine (68a)and (R)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(68b)

Compound N-(butan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(26.7 mg) (Example 68) was subjected to chiral separation to get twoenantiomers, peak 1 (T_(R)=1.44 min) isomer (11.7 mg) and peak 2(T_(R)=1.94 min) isomer 11.6 mg. Chiral center assignments aretentative. Chiral separation conditions: solvent A CO₂(85%), solvent BMeOH(15%), flow rate 2 ml/min, temp 30° C., column 21×250 mm AD-H, runtime 3.5 minute stacked injections, 7 minute elution time.

Peak 1 (T_(R)=1.44 min) isomer: 1H NMR (500 MHz, DMSO-d6) δ 9.18 (s,1H), 8.77 (d, J=5.5 Hz, 2H), 8.65 (d, J=5.6 Hz, 1H), 8.43 (d, J=7.8 Hz,1H), 8.36-8.31 (m, 2H), 8.29 (d, J=5.6 Hz, 1H), 4.56 (p, J=7.2 Hz, 1H),1.85-1.62 (m, 2H), 1.33 (d, J=6.6 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H). LCMS(m/z [M+H]⁺): 280.2.

Peak 2 (T_(R)=1.94 min) isomer: 1H NMR (500 MHz, DMSO-d6) δ 9.18 (s,1H), 8.80-8.72 (m, 2H), 8.65 (d, J=5.6 Hz, 1H), 8.43 (d, J=7.8 Hz, 1H),8.35-8.31 (m, 2H), 8.30 (dd, J=5.6, 0.8 Hz, 1H), 4.57 (hept, J=6.7 Hz,1H), 1.82-1.63 (m, 2H), 1.33 (d, J=6.6 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H).LCMS (m/z [M+H]⁺): 280.2.

Example 69:N-(2-methylbut-3-yn-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.25 (d, J=0.9 Hz, 1H), 8.77-8.71 (m,2H), 8.64-8.58 (m, 3H), 8.23 (dd, J=5.7, 0.9 Hz, 1H), 2.81 (s, 1H), 1.93(s, 6H). LCMS (m/z [M+H]⁺): 290.1.

Example 70:(1r,3s)-3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutan-1-ol

1H NMR (400 MHz, DMSO-d6) 9.21 (s, 1H), 8.78 (d, 2H), 8.55 (d, 1H), 8.31(d, 2H), 8.05 (d, 1H), 3.94(q, 2H), 3.50 (s, 3H), 1.39 (t, 3H). LCMS(m/z [M+H]⁺): 308.1.

Example 71:2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol

1H NMR (500 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.82-8.77 (m, 2H),8.67 (d, J=5.6 Hz, 1H), 8.28-8.23 (m, 2H), 8.07 (dd, J=5.8, 0.9 Hz, 1H),7.49 (s, 1H), 1.64 (s, 6H), 1.27 (s, 6H). LCMS (m/z [M+H]⁺): 324.2.

Example 72:2-(pyridin-4-yl)-N-(2,4,4-trimethylpentan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.19 (d, J=0.9 Hz, 1H), 8.78-8.72 (m,2H), 8.58 (d, J=5.7 Hz, 1H), 8.50-8.45 (m, 2H), 8.22 (dd, J=5.8, 0.9 Hz,1H), 2.33 (d, J=1.2 Hz, 2H), 1.77 (s, 6H), 1.01 (s, 9H). LCMS (m/z[M+H]⁺): 336.2.

Example 73:N-(pentan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.79-8.74 (m, 2H),8.65 (d, J=5.6 Hz, 1H), 8.37-8.30 (m, 4H), 4.47 (dtd, J=13.2, 8.2, 5.1Hz, 1H), 1.83-1.63 (m, 4H), 0.95 (t, J=7.4 Hz, 6H). LCMS (m/z [M+H]⁺):294.2.

Example 74:(N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.83-8.77 (m, 2H),8.65 (d, J=5.6 Hz, 1H), 8.35 (dd, J=5.8, 0.9 Hz, 1H), 8.32-8.28 (m, 2H),7.79 (s, 1H), 3.55-3.49 (m, 4H), 2.99 (s, 2H), 2.51-2.45 (m, 4H), 1.62(s, 6H). LCMS (m/z [M+H]⁺): 365.2.

Example 75:N-[1-(tert-butoxy)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.82-8.77 (m, 2H),8.65 (d, J=5.6 Hz, 1H), 8.38 (dd, J=5.7, 0.9 Hz, 1H), 8.33-8.28 (m, 2H),7.66 (s, 1H), 3.84 (s, 2H), 1.60 (s, 6H), 1.05 (s, 9H). LCMS (m/z[M+H]⁺): 352.2.

Example 76:4,4,4-trifluoro-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol

1H NMR (500 MHz, DMSO-d6) δ 9.23 (d, J=0.8 Hz, 1H), 8.81-8.76 (m, 2H),8.70 (d, J=5.5 Hz, 1H), 8.62 (d, J=8.2 Hz, 1H), 8.36-8.31 (m, 2H), 8.22(dd, J=5.7, 0.9 Hz, 1H), 5.19 (dd, J=6.3, 5.4 Hz, 1H), 4.98 (tq, J=9.6,5.9 Hz, 1H), 3.72 (dt, J=10.9, 5.5 Hz, 1H), 3.61 (dt, J=11.0, 6.3 Hz,1H), 2.87-2.71 (m, 2H). LCMS (m/z [M+H]⁺): 350.1.

Example 77: N-pentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.83 (t, J=5.6 Hz,1H), 8.79-8.73 (m, 2H), 8.64 (d, J=5.5 Hz, 1H), 8.36-8.29 (m, 2H), 8.18(dd, J=5.6, 1.0 Hz, 1H), 3.70 (td, J=7.2, 5.6 Hz, 2H), 1.81-1.68 (m,2H), 1.47-1.30 (m, 4H), 0.93-0.86 (m, 3H). LCMS (m/z [M+H]⁺): 294.2.

Example 78:2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.80-8.74 (m, 2H),8.65 (d, J=5.6 Hz, 1H), 8.40-8.30 (m, 4H), 4.83 (t, J=5.7 Hz, 1H), 4.54(td, J=8.4, 4.9 Hz, 1H), 3.72-3.56 (m, 2H), 1.84 (ddd, J=14.0, 7.4, 5.1Hz, 1H), 1.68 (ddd, J=13.8, 8.8, 7.3 Hz, 1H), 0.96 (t, J=7.4 Hz, 3H).LCMS (m/z [M+H]⁺): 296.1.

Example 79:N-[1-(1H-indol-3-yl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 10.78 (d, J=2.6 Hz, 1H), 9.21 (d, J=0.8 Hz,1H), 8.86-8.78 (m, 2H), 8.60 (d, J=5.6 Hz, 1H), 8.42-8.36 (m, 2H), 8.30(dd, J=5.7, 0.9 Hz, 1H), 7.68 (s, 1H), 7.47-7.40 (m, 1H), 7.29 (dt,J=8.2, 0.9 Hz, 1H), 7.00 (ddd, J=8.1, 7.0, 1.1 Hz, 1H), 6.91-6.82 (m,2H), 3.59 (s, 2H), 1.66 (s, 6H). LCMS (m/z [M+H]⁺): 395.2.

Example 80:N-[1-(4-fluorophenyl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.23 (d, J=0.9 Hz, 1H), 8.80-8.75 (m,2H), 8.58-8.51 (m, 3H), 8.13 (dd, J=5.7, 0.9 Hz, 1H), 7.11-7.04 (m, 2H),6.95-6.87 (m, 2H), 3.56 (s, 2H), 1.69 (s, 6H). LCMS (m/z [M+H]⁺): 374.2.

Example 81:N-(2-phenylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.16 (d, J=0.9 Hz, 1H), 8.69 (d, J=5.6 Hz,1H), 8.66 (s, 1H), 8.61-8.56 (m, 2H), 8.53 (dd, J=5.6, 0.9 Hz, 1H),7.80-7.75 (m, 2H), 7.55-7.48 (m, 2H), 7.35-7.28 (m, 2H), 7.20-7.12 (m,1H), 1.89 (s, 6H). LCMS (m/z [M+H]⁺): 342.2.

Example 82:N-(2-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.33 (d, J=1.0 Hz, 1H), 8.71 (d, J=5.7Hz, 1H), 8.68-8.64 (m, 2H), 8.32 (ddd, J=9.5, 5.1, 1.3 Hz, 3H), 7.81(td, J=8.0, 2.0 Hz, 1H), 7.41 (d, J=1.1 Hz, 1H), 7.38-7.31 (m, 2H). LCMS(m/z [M+H]⁺): 318.1.

Example 83:N-[2-(4-fluorophenyl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.18 (d, J=0.9 Hz, 1H), 8.63 (d, J=5.7Hz, 1H), 8.59-8.53 (m, 2H), 8.36 (dd, J=5.7, 0.9 Hz, 1H), 7.97-7.92 (m,2H), 7.61-7.53 (m, 2H), 7.11-7.02 (m, 2H), 1.96 (s, 6H). LCMS (m/z[M+H]⁺): 360.2.

Example 84:3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid

1H NMR (500 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.24 (d, J=0.8 Hz, 1H),8.81-8.75 (m, 2H), 8.71 (d, J=5.5 Hz, 1H), 8.34-8.29 (m, 2H), 7.84 (d,J=5.5 Hz, 1H), 7.07 (s, 1H), 2.03 (s, 3H). LCMS (m/z [M+H]⁺): 364.1.

Example 85:2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.88 (t, J=5.3 Hz,1H), 8.82-8.73 (m, 2H), 8.65 (d, J=5.6 Hz, 1H), 8.38-8.31 (m, 2H), 8.21(dd, J=5.6, 0.9 Hz, 1H), 4.90 (t, J=5.3 Hz, 1H), 3.76 (ttd, J=7.9, 5.5,2.5 Hz, 4H). LCMS (m/z [M+H]⁺): 268.1.

Example 86: N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.9 Hz, 1H), 8.88 (q, J=4.4 Hz,1H), 8.80-8.73 (m, 2H), 8.65 (d, J=5.5 Hz, 1H), 8.40-8.33 (m, 2H), 8.12(dd, J=5.6, 1.0 Hz, 1H), 3.18 (d, J=4.5 Hz, 3H). LCMS (m/z [M+H]⁺):238.1.

Example 87:1-({[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}methyl)cyclopentan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.81-8.74 (m, 2H),8.67 (dd, J=11.8, 5.8 Hz, 2H), 8.37-8.33 (m, 2H), 8.30 (dd, J=5.6, 0.9Hz, 1H), 4.70 (s, 1H), 3.89 (d, J=5.9 Hz, 2H), 1.78-1.50 (m, 8H). LCMS(m/z [M+H]⁺): 322.2.

Example 88: N,N-dimethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J=0.8 Hz, 1H), 8.80-8.73 (m, 2H),8.57 (d, J=5.8 Hz, 1H), 8.38-8.31 (m, 2H), 8.15 (dd, J=5.8, 0.8 Hz, 1H),3.53 (s, 6H). LCMS (m/z [M+H]⁺): 252.1.

Example 89:N-(2-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 10.22 (s, 1H), 9.29 (d, J=0.9 Hz, 1H), 8.76(d, J=5.5 Hz, 1H), 8.72-8.67 (m, 2H), 8.42 (dd, J=5.7, 1.0 Hz, 1H),8.09-8.04 (m, 2H), 7.50 (dd, J=7.7, 1.5 Hz, 1H), 7.43 (dd, J=7.1, 1.8Hz, 1H), 7.40-7.29 (m, 2H), 2.27 (s, 3H). LCMS (m/z [M+H]⁺): 314.1.

Example 90:N-(4-methylphenyl)-2-(pyridin-4-yl)pyridol3,4-dWrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.29 (d, J=0.8 Hz, 1H),8.81-8.73 (m, 3H), 8.49 (dd, J=5.7, 1.0 Hz, 1H), 8.30-8.25 (m, 2H),7.87-7.80 (m, 2H), 7.36-7.30 (m, 2H), 2.38 (s, 3H). LCMS (m/z [M+H]⁺):314.1.

Example 91:N-(4-methoxyphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.27 (d, J=0.8 Hz, 1H),8.80-8.72 (m, 3H), 8.46 (dd, J=5.8, 1.0 Hz, 1H), 8.29-8.23 (m, 2H),7.88-7.79 (m, 2H), 7.14-7.05 (m, 2H), 3.83 (s, 3H). LCMS (m/z [M+H]⁺):330.1.

Example 92: N-phenyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.30 (d, J=0.8 Hz, 1H),8.82-8.74 (m, 3H), 8.51 (dd, J=5.8, 1.0 Hz, 1H), 8.31-8.24 (m, 2H),8.00-7.92 (m, 2H), 7.58-7.48 (m, 2H), 7.26 (tt, J=7.3, 1.2 Hz, 1H). LCMS(m/z [M+H]⁺): 300.1.

Example 93:N-(3-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.30 (d, J=0.8 Hz, 1H),8.82-8.75 (m, 3H), 8.51 (dd, J=5.8, 0.9 Hz, 1H), 8.31-8.26 (m, 2H),7.83-7.76 (m, 2H), 7.41 (t, J=7.8 Hz, 1H), 7.08 (ddt, J=7.6, 1.7, 0.9Hz, 1H), 2.43 (s, 3H). LCMS (m/z [M+H]⁺): 314.1.

Example 94:6-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}hexanoic acid

1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 9.19 (s, 1H), 8.84 (t, J=5.6Hz, 1H), 8.80-8.73 (m, 2H), 8.65 (d, J=5.6 Hz, 1H), 8.37-8.31 (m, 2H),8.18 (dd, J=5.6, 1.0 Hz, 1H), 3.76-3.66 (m, 2H), 2.23 (t, J=7.3 Hz, 2H),1.81-1.69 (m, 2H), 1.61 (p, J=7.3 Hz, 2H), 1.43 (tt, J=9.6, 6.1 Hz, 2H).LCMS (m/z [M+H]⁺): 338.2.

Example 95:N-(3-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.34 (d, J=0.8 Hz, 1H),8.84-8.78 (m, 3H), 8.51 (dd, J=5.8, 0.9 Hz, 1H), 8.31-8.26 (m, 2H), 7.96(dt, J=11.7, 2.3 Hz, 1H), 7.81 (ddd, J=8.2, 2.0, 0.9 Hz, 1H), 7.56 (td,J=8.2, 6.8 Hz, 1H), 7.08 (tdd, J=8.5, 2.6, 0.9 Hz, 1H). LCMS (m/z[M+H]⁺): 318.1.

Example 96:N-(4-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.31 (d, J=0.9 Hz, 1H),8.81-8.75 (m, 3H), 8.47 (dd, J=5.7, 0.9 Hz, 1H), 8.31-8.24 (m, 2H),7.99-7.92 (m, 2H), 7.41-7.33 (m, 2H). LCMS (m/z [M+H]⁺): 318.1.

Example 97:4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoic acid

1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.16 (s, 1H), 8.79-8.72 (m,2H), 8.62 (d, J=5.5 Hz, 1H), 8.39-8.32 (m, 2H), 8.19 (dd, J=5.6, 1.0 Hz,1H), 3.67 (q, J=6.0 Hz, 2H), 2.33 (t, J=6.5 Hz, 2H), 1.94 (p, J=6.6 Hz,2H). LCMS (m/z [M+H]⁺): 365.2.

Example 98:N-(1-phenylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (DMSO-d6) δ 1.67 (d, J=6.8 Hz, 3H), 5.73 (m, 1H), 7.22 (m, 1H),7.47 (m, 2H), 7.55 (m, 2H), 8.26 (d, J=5 Hz, 2H), 8.41 (d, J=5.6 Hz,1H), 8.69 (d, J=5.6 Hz, 1H), 8.74 (d, J=5 Hz, 1H), 9.08 (d, J=7.2 Hz,1H), 9.19 (s, 1H). LCMS (m/z [M+H]⁺): 328.2.

Example 99:N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.19 (s, 1H), 8.73 (d, J=5.7 Hz, 2H),8.55 (m, 3H), 8.01 (dd, J=5.7, 0.7 Hz, 1H), 1.64 (s, 3H), 0.99 (m, 2H),0.93 (m, 2H). LCMS (m/z [M+H]⁺): 278.1.

Example 100: Tert-butylN-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)carbamate

1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 8.87 (s, 2H), 8.67 (d, J=5.6Hz, 1H), 8.49 (d, J=4.6 Hz, 2H), 8.24 (d, J=5.6 Hz, 1H), 8.03 (s, 1H),7.33 (t, J=6.4 Hz, 1H), 3.54 (d, J=6.4 Hz, 2H), 1.57 (s, 6H), 1.36 (s,9H). LCMS (m/z [M+H]⁺): 395.2.

Example 101:(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol

1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.78-8.72 (m, 2H), 8.68 (s,1H), 8.62 (d, J=5.6 Hz, 1H), 8.36 (d, J=5.7 Hz, 1H), 8.31-8.20 (m, 2H),4.94 (t, J=6.0 Hz, 1H), 3.96 (d, J=6.0 Hz, 2H), 2.41 (dt, J=12.5, 7.2Hz, 4H), 1.89 (p, J=8.0 Hz, 2H). LCMS (m/z [M+H]⁺): 308.1.

Example 102: methyl2-(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)acetate

1H NMR (400 MHz, DMSO-d6) 9.25 (s, 1H), 9.05 (t, 1H), 8.90(d, 2H), 6.70(d, 1H), 8.60 (d, 2H), 8.24 (d, 1H), 3.90 (m, 2H), 3.79 (t, 2H), 3.59(m, 2H), 3.55-3.49 (m, 4H), 3.45 (m, 2H), 3.41 (m, 2H), 2.40 (t, 2H).LCMS (m/z [M+H]⁺): 336.1.

Example 103:N-(2-methylpropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1HNMR (400 MHz, CDCl₃) δ 9.35 (d, J=0.8 Hz, 1H), 8.79 (d, J=5.6 Hz, 2H),8.64 (d, J=5.6 Hz, 1H), 8.39 (d, J=5.6 Hz, 2H), 7.52 (dd, J=5.6, 0.8 Hz,1H), 6.00 (t, J=6.0 Hz, 1H), 3.66 (dd, J=6.8, 6.0 Hz, 2H), 2.09 (nonet,J=6.8 Hz, 1H), 1.12 (d, J=6.8 Hz, 6H). LCMS (m/z [M+H]⁺): 280.1.

Example 104:3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanenitrile

1H NMR (500 MHz, Acetone-d6) δ 9.23 (d, J=0.9 Hz, 1H), 8.82-8.75 (m,2H), 8.63 (d, J=5.6 Hz, 1H), 8.42-8.33 (m, 2H), 8.17 (dd, J=5.7, 0.9 Hz,1H), 3.64 (s, 2H), 1.83 (s, 6H). LCMS (M/Z [M+H]⁺): 305.1.

Example 105:N-(6-aminohexyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.88-8.80 (m, 1H), 8.82-8.73(m, 2H), 8.68-8.61 (m, 1H), 8.37-8.31 (m, 2H), 8.19 (d, J=5.6 Hz, 1H),3.71 (q, J=6.5 Hz, 2H), 2.53 (d, J=1.6 Hz, 2H), 1.75 (dt, J=14.2, 7.1Hz, 2H), 1.47-1.32 (m, 6H). LCMS (M/Z [M+H]⁺): 323.2.

Example 106:N-(4-aminobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.96 (t, J=5.7 Hz, 1H),8.88-8.82 (m, 2H), 8.70 (d, J=5.5 Hz, 1H), 8.51-8.44 (m, 2H), 8.20 (dd,J=5.6, 1.0 Hz, 1H), 7.65 (s, 2H), 3.80-3.72 (m, 2H), 2.86 (td, J=7.5,5.5 Hz, 2H), 1.85-1.73 (m, 2H), 1.67 (ddt, J=12.8, 9.9, 5.8 Hz, 2H).LCMS (M/Z [M+H]⁺): 295.2.

Example 107:2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanenitrile

1H NMR (500 MHz, DMSO-d6) δ 9.31 (d, J=0.8 Hz, 1H), 8.85-8.80 (m, 2H),8.78-8.72 (m, 2H), 8.45-8.40 (m, 2H), 8.37 (dd, J=5.7, 0.9 Hz, 1H), 1.93(s, 6H). LCMS (M/Z [M+H]⁺): 291.1.

Example 108:N-[2-methyl-1-(2-methylpiperidin-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.08 (d, J=0.9 Hz, 1H), 8.65-8.58 (m,2H), 8.50 (d, J=5.7 Hz, 1H), 8.37-8.29 (m, 2H), 7.94 (dd, J=5.8, 1.0 Hz,1H), 3.18 (d, J=14.7 Hz, 1H), 3.00-2.92 (m, 2H), 2.66 (s, 1H), 2.44 (dq,J=12.3, 5.9, 5.1 Hz, 1H), 1.86 (s, 1H), 1.69-1.43 (m, 10H), 1.38-1.25(m, 2H), 0.97 (d, J=6.4 Hz, 3H). LCMS (M/Z [M+H]⁺): 377.2.

Example 109:dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine

1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.96 (t, J=5.7 Hz, 1H),8.88-8.82 (m, 2H), 8.70 (d, J=5.5 Hz, 1H), 8.51-8.44 (m, 2H), 8.20 (dd,J=5.6, 1.0 Hz, 1H), 7.65 (s, 2H), 3.80-3.72 (m, 2H), 2.86 (td, J=7.5,5.5 Hz, 2H), 1.85-1.73 (m, 2H), 1.67 (ddt, J=12.8, 9.9, 5.8 Hz, 2H).LCMS (M/Z [M+H]⁺): 295.2.

Example 110:N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.88 (s, 2H), 8.72 (d, J=5.6Hz, 1H), 8.45 (d, J=5.7 Hz, 1H), 8.39 (d, J=4.9 Hz, 2H), 7.87 (s, 1H),7.79 (s, 2H), 3.67 (d, J=5.9 Hz, 2H), 1.64 (s, 6H). LCMS (M/Z [M+H]⁺):295.2.

Example 111:N-cyclopentyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine

The title compound was prepared from 2,4-dichloropyrido[3,4-d]pyrimidine(intermediate 2c) as in Scheme 2 using Step C for Example 1 and Step Afor Intermediate 6b. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J=0.8 Hz, 1H),8.60 (d, J=5.6 Hz, 1H), 8.36 (dd, J=5.8, 0.9 Hz, 1H), 8.09 (dd, J=5.3,0.8 Hz, 1H), 7.77 (s, 1H), 7.50 (dd, J=1.5, 0.8 Hz, 1H), 7.42 (dd,J=5.3, 1.5 Hz, 1H), 6.10 (s, 2H), 1.60 (s, 9H). LCMS (m/z [M+H]⁺):349.1.

Examples 112-197

These compounds were synthesized according to the protocol describedabove using 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andvarious amines and coupling partners respectively except speciallystated.

Example 112:4-[4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl]pyridin-2-amine

1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J=0.8 Hz, 1H), 8.60 (d, J=5.6 Hz,1H), 8.36 (dd, J=5.8, 0.9 Hz, 1H), 8.09 (dd, J=5.3, 0.8 Hz, 1H), 7.77(s, 1H), 7.50 (dd, J=1.5, 0.8 Hz, 1H), 7.42 (dd, J=5.3, 1.5 Hz, 1H),6.10 (s, 2H), 1.60 (s, 9H). LCMS (m/z [M+H]⁺): 295.2

Example 113:2-[1-(benzenesulfonyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-N-tert-butylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J=0.8 Hz, 1H), 8.70(dd, J=7.9, 1.7HZ, 1H), 8.58 (d, J=5.6 Hz, 1H), 8.20 (m, 2H), 7.71 (m, 1H), 7.66-7.59(m, 5H), 7.35 (dd, J=7.9, 4.7 Hz, 1H), 3.24 (s, 3H), 1.59 (s, 9H). LCMS(m/z [M+H]⁺): 473.2.

Example 114:N-tert-butyl-2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine

The title compound was prepared by de-protection of the tosyl group ofExample 113.

De-protection:N-(tert-butyl)-2-(2-methyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrido[3,4-d]pyrimidin-4-amine(32mg, 0.068 mmol) was stirred in 1 mL of t-butanol at room temperature.Sodium hydroxide (270 microlitre (5M), 1.35 mmol) was then added and thereaction stirred at room temperature for two hours upon which time allstarting material was consumed. The material was concentrated to an offwhite oil and purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-20% MeOH/DCM to afford the title productN-tert-butyl-2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine(55%).

1H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 1H), 9.05 (d, J=0.8 Hz, 1H), 8.97(dd, J=8.0, 1.7 Hz, 1H), 8.45 (d, J=5.5 Hz, 1H), 8.25(ddd, J=23.7, 5.2,1.3 Hz, 1H), 8.19 (m, 1H), 7.42 (s, 1H), 7.15 (dd, J=7.9, 4.7 Hz, 1H),2.96 (s, 3H), 1.63 (s, 9H). LCMS (m/z [M+H]⁺): 333.2.

Example 115:N-tert-butyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 8.95 (d, J=0.8 Hz, 1H), 8.54 (d, J=5.6 Hz,1H), 8.46 (s, 1H), 8.30 (dd, J=5.7, 0.9 Hz, 1H), 7.62 (s, 1H), 1.58 (s,9H). LCMS (m/z [M+H]⁺): 337.1.

Example 116:N-tert-butyl-2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J=0.8 Hz, 1H), 8.79 (d, J=0.5 Hz,1H), 8.68 (m, 2H), 8.41 (dd, J=5.8, 0.9 Hz, 1H), 7.92 (s, 1H), 7.77 (dd,J=4.9, 0.6 Hz, 1H), 1.55 (s, 9H). LCMS (m/z [M+H]⁺): 314.1.

Example 117:N-tert-butyl-2-(3-methylpyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.11 (d, J=0.8 Hz, 1H), 8.63 (d, J=5.6 Hz,1H), 8.55 (m, 2H), 8.39 (dd, J=5.7, 0.9 Hz, 1H), 7.79 (m, 1H), 7.76 (m,1H), 2.59 (s, 3H), 1.58 (s, 9H). LCMS (m/z [M+H]⁺): 294.2.

Example 118:2-(3-chloropyridin-4-yl)-N-(2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J=0.9 Hz, 1H), 8.78 (d, J=0.6 Hz,1H), 8.68 (dd, J=5.3, 2.2 Hz, 1H), 8.66(m, 2H), 8.41 (dd, J=5.7, 0.9 Hz,1H), 7.74 (m, 1H), 2.00(q, J=7.3 Hz, 2H), 1.49 (s, 6H), 0.80 (t, J=7.4Hz, 3H). LCMS (m/z [M+H]⁺): 328.1.

Example 119:2,4-dimethyl-4-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)pentan-2-ol

1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.99 (d, J=0.8 Hz, 1H), 8.55(d, J=5.6 Hz, 1H), 8.49 (s, 1H), 7.73 (dd, J=5.7, 0.9 Hz, 1H), 5.61 (s,1H), 1.97 (s, 2H), 1.70 (s, 6H), 1.30 (s, 6H). LCMS (m/z [M+H]⁺): 395.2.

Example 120:N-ethyl-2-(3-fluoropyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared from 2,4-dichloropyrido[3,4-d]pyrimidine(intermediate 2c) similar as described in Example 111 except usingStille coupling as the second step.

Stille coupling:2-chloro-N-ethyl-N-isopropylpyrido[3,4-d]pyrimidin-4-amine (30 mg, 0.12mmol) was stirred in dry DMF (1 mL) at room temperature.3-fluoro-4-(tributylstannyl)pyridine (50.8 mg, 0.13 mmol) was added thenPdCl2(dppf). CH2Cl2 adduct(9.8 mg, 0.012 mmol) and Cul(2.3 mg, 0.012mmol). The reaction was stirred for 1 hour at 130° C. The reaction wasthen concentrated and purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-10% MeOH/DCM to afford the title compound.

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.72 (d, J=2.9 Hz,1H), 8.61 (m, 1H), 8.59 (m, 1H), 8.08 (dd, J=6.7, 4.9 Hz, 1H), 7.90 (m,1H), 4.92-4.88 (m, 1H), 3.79(q, J=7.0 Hz, 2H), 1.37 (m, 6H), 1.32 (dd,J=23.7, 6.8 Hz, 3H). LCMS (m/z [M+H]⁺): 312.2.

Example 121:2-methyl-1-[2-methyl-2-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)propoxy]propan-2-ol

The title compound was prepared using procedures described in Example111 and the amine as described in Example 12.

1H NMR (400 MHz, DMSO-d6) δ 13.80 (s, 1H), 8.99 (d, J=0.8 Hz, 1H), 8.55(d, J=5.6 Hz, 1H), 8.44 (s, 1H), 8.25 (dd, J=5.8, 0.9 Hz, 1H), 7.53 (s,1H), 4.35 (s, 1H), 3.82 (s, 2H), 3.17 (s, 2H), 1.54 (s, 6H), 1.00 (s,6H). LCMS (m/z [M+H]⁺): 425.2.

Example 122:2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

The title compound was prepared using procedures described in Example120.

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.8 Hz, 1H), 8.74 (d, J=2.9 Hz,1H), 8.60 (m, 2H), 8.11 (m, 1H), 8.09 (m, 1H), 5.11-5.06 (d, J=6.6 Hz,1H), 3.37 (s, 3H), 1.34 (d, J=6.7 Hz, 6H). LCMS (m/z [M+H]⁺): 298.1.

Example 123:N-ethyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.59 (m, 1H), 8.56(m, 1H), 8.54 (m, 1H), 7.89 (m, 1H), 7.85 (m, 1H), 4.95-4.90 (d, J=6.6Hz, 1H), 3.79(q, J=7.0 Hz, 2H), 2.60 (s, 3H), 1.36 (d, J=6.6 Hz, 6H),1.30 (t, J=7.0 Hz, 3H). LCMS (m/z [M+H]⁺): 308.2.

Example 124:2-(3-chloropyridin-4-yl)-N-(1-methoxy-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.79 (s, 1H), 8.69 (dd, J=5.2,1.8 Hz, 2H), 8.40 (dd, J=5.8, 1.0 Hz, 1H), 7.78 (s, 1H), 7.75 (m, 1H),3.73 (s, 2H), 3.22 (s, 3H), 1.50 (s, 6H). LCMS (m/z [M+H]⁺): 344.1.

Example 125:4-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol

1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 9.13 (d, J=0.7 Hz, 1H), 8.79(s, 1H), 8.68(dd, J=5.3, 3.4 z, 1H), 8.66 (m, 1H), 7.83 (dd, J=5.7, 0.9Hz, 1H), 7.77 (dd, J=4.9, 0.6 Hz, 1H), 5.57 (s, 1H), 1.96 (s, 2H), 1.65(s, 6H), 1.29 (s, 6H). LCMS (m/z [M+H]⁺): 372.2.

Example 126:2-(3-chloropyridin-4-yl)-N-cyclopentylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.14 (d, J=0.9 Hz, 1H), 8.78 (d, J=0.6 Hz,1H), 8.69 (dd, J=9.4, 5.2 Hz, 1H), 8.67 (d, J=6.9 Hz, 1H), 8.61(d, J=6.8Hz, 1H), 8.32 (dd, J=5.7, 0.9 Hz, 1H), 7.81 (m, 1H), 4.61-4.57 (m, 1H),2.10-2.01 (m, 2H), 1.80-1.54 (m, 6H). LCMS (m/z [M+H]⁺): 326.1.

Example 127:1-(2-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2-methylpropoxy)-2-methylpropan-2-ol

The title compound was prepared using procedures described in Example111 and the amine as described in Example 12.

1H NMR (400 MHz, DMSO-d6) δ 9.13 (m, 1H), 8.80 (d, J=0.6 Hz, 1H), 8.69(m, 2H), 8.40 (dd, J=5.8, 0.9 Hz, 1H), 7.83 (dd, J=4.9, 0.6 Hz, 1H),7.76 (dd, J=4.9, 0.6 Hz, 1H), 4.38 (s, 1H), 3.80 (s, 2H), 3.16 (s, 2H),1.51 (s, 6H), 1.00 (s, 6H). LCMS (m/z [M+H]⁺): 402.2.

Example 128:N-methyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J=0.8 Hz, 1H), 8.58 (m, 2H), 8.53(m, 1H), 8.03 (dd, J=5.8, 0.9 Hz, 1H), 7.87 (d, J=5.0 Hz, 1H), 5.09-5.00(m, 1H), 3.29 (s, 3H), 2.60 (s, 3H), 1.31 (d, J=6.7 Hz, 6H). LCMS (m/z[M+H]⁺): 294.2.

Example 129:2-(3-chloropyridin-4-yl)-N-(4-methanesulfonyl-2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J=0.8 Hz, 1H), 8.79 (d, J=0.6 Hz,1H), 8.69 (dd, J=21.3, 5.3 Hz, 1H), 8.65(m, 1H), 8.40 (dd, J=5.8, 0.9Hz, 1H), 7.80 (m, 1H), 7.78 (m, 1H), 3.12-3.08 (m, 2H), 2.87 (s, 3H),2.49-2.46 (m, 2H), 1.53 (s, 6H). LCMS (m/z [M+H]⁺): 406.1.

Example 130:N-tert-butyl-2-[3-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 8.92 (m, 1H), 8.89 (m, 1H), 8.08 (d, J=4.9Hz, 1H), 7.76 (d, J=0.8 Hz, 1H), 7.70 (m, 1H), 7.67 (m, 1H), 7.56 (dd,J=5.0, 0.8 Hz, 1H), 1.49 (s, 9H). LCMS (m/z [M+H]⁺): 348.1.

Example 131:N-tert-butyl-2-[2-chloro-5-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.08 (d, J=0.9 Hz, 1H), 8.87 (s, 1H), 8.61(d, J=5.8 Hz, 1H), 8.22 (dd, J=5.8, 1.0 Hz, 1H), 7.83 (s, 1H), 1.20 (s,9H). LCMS (m/z [M+H]⁺): 382.1.

Example 132:2-(3-chloropyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.15 (d, J=0.8 Hz, 1H), 8.77(s, 1H), 8.70 (dd, J=18.4, 5.2 Hz, 1H), 8.65 (m, 1H), 8.25 (dd, J=5.7,0.9 Hz, 1H), 7.81 (d, J=4.9 Hz, 1H), 3.70-3.65(td, J=6.8, 5.1 Hz, 2H),2.90 (t, J=7.4 Hz, 2H), 2.12-2.07 (m, 2H). LCMS (m/z [M+H]+): 368.1.

Example 133:2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.41 (d, J=5.7 Hz, 1H),8.26 (s, 1H), 7.91 (dd, J=5.7, 0.9 Hz, 1H), 2.83 (s, 3H), 1.60 (s, 3H),1.05-0.94 (m, 2H), 0.91-0.82 (m, 2H). LCMS (m/z [M+H]⁺): 281.1.

Example 134:2-(3-fluoropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared using procedures described in Example 120.

1H NMR (600 MHz, DMSO-d6) δ 9.24 (d, J=0.8 Hz, 1H), 8.77-8.73 (m, 2H),8.62 (dd, J=4.9, 0.8 Hz, 1H), 8.52 (dd, J=5.8, 0.9 Hz, 1H), 8.04 (dd,J=6.7, 4.9 Hz, 1H), 7.99 (s, 1H), 1.86 (s, 6H). LCMS (m/z [M+H]⁺):352.1.

Example 135:2-(3-methyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (600 MHz, Methanol-d4) δ 9.06 (d, J=0.9 Hz, 1H), 8.47 (d, J=5.7Hz, 1H), 8.16 (dd, J=5.7, 0.9 Hz, 1H), 8.11 (s, 1H), 2.74 (d, J=34.7 Hz,3H), 1.92 (s, 6H). LCMS (m/z [M+H]⁺): 337.1.

Example 136:2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine

Step 1: A mixture of urea (40.00 g, 666.00 mmol) and 3-aminoisonicotinicacid (2a, 18.40 g, 133.20 mmol) was heated at 210° C. for 1 hr (NOTE: nosolvent was used). NaOH (2N, 320 mL) was added, and the mixture wasstirred at 90° C. for 1 h. The solid was collected by filtration, andwashed with water. The crude product thus obtained was suspended in HOAc(400 mL), and stirred at 100° C. for 1 h. The mixture was cooled to RT,filtered, and the solid was washed with a large amount of water, andthen dried under the vacuum to givepyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b, 17.00 g, 78% yield)without further purification. LCMS (m/z [M+H]⁺): 164.0.

Step 2: To a mixture of pyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (2b,20.00 g, 122.60 mmol) and POCl₃ (328.03 g, 2.14 mol) in toluene (200 mL)was added DIEA (31.69 g, 245.20 mmol) dropwise and this reaction mixturestirred at 25° C. overnight (18 hr) to give suspension.

The solvent and POCl₃ was removed under vacuum, diluted with DCM (50mL), neutralized with DIEA to pH=7 at −20° C. and concentrated again,the residue was purified by column (20-50% EA/PE) to give2,4-dichloropyrido[3,4-d]pyrimidine (2c, 20.00 g, 99.99 mmol, 82% yield)as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 9.52 (s, 1H), 8.92(d, J=5.6 Hz, 1H), 8.04 (d, J=5.6 Hz, 1H). LCMS (m/z [M+H]⁺): 200.0.

Step 3: In a 20 mL vial 2,4-dichloropyrido[3,4-d]pyrimidine (600 mg, 3.0mmol) was stirred in DMSO (0.7 mL) at room temperature and degassed withN₂. DIEA (1 mL, 6 mmol) was added and stirred for 5 minutes then KF (174mg, 3 mmol). This mixture was stirred at room temperature for 15 minutesthen racemic 1,1,1-trifluoro-N-methylpropan-2-amine (419 mg, 3.3 mmol)was added and degassed then stirred at 60° C. for 4 hours. The reactionwas then concentrated and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to afford2-chloro-N-methyl-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine(680 mg, 74%). 1H NMR (500 MHz, Acetone-d6) δ 9.09 (d, J=0.9 Hz, 1H),8.59 (d, J=5.9 Hz, 1H), 8.22 (dd, J=5.9, 0.9 Hz, 1H), 5.93 (dddd,J=15.3, 8.3, 7.0, 1.2 Hz, 1H), 3.61 (q, J=1.0 Hz, 3H), 1.63 (d, J=7.0Hz, 3H). LCMS (m/z [M+H]⁺): 291.7.

Step 4: In a 20 mL vial2-chloro-N-methyl-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine(100 mg, 0.34 mmol) was stirred in dry DMF (1 mL) at room temperature.3-fluoro-4-(tributylstannyl)pyridine (133 mg, 0.34 mmol) was added thenPdCl₂(dppf). CH₂Cl₂ adduct (28.1 mg, 0.034 mmol) and Cul (6.55 mg, 0.034mmol). The reaction was stirred for 0.5 hour at 130° C. The crudemixture was diluted with DCM, H₂O, separated and extracted with DCM×3.Combined the organic layers and dried Na₂SO₄, filtered and concentrated.The residue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-10% MeOH/DCM to give the racemic product, then followedby chiral HPLC (21×250 mm OJ-H column with 85% CO₂ as phase A and 15%MeOH as phase B, flow rate 2 mL/min, 30° C., 2.75 min elution time) toseparate the enantiomers to afford Examples 136 and 137.

Examples 136: 1H NMR (500 MHz, DMSO-d6) δ 9.30 (d, J=0.8 Hz, 1H), 8.75(d, J=3.0 Hz, 1H), 8.66 (d, J=5.9 Hz, 1H), 8.62 (dd, J=4.9, 0.8 Hz, 1H),8.24 (dd, J=5.9, 0.9 Hz, 1H), 8.15 (dd, J=6.8, 4.9 Hz, 1H), 6.12-5.98(m, 1H), 3.51 (d, J=1.1 Hz, 3H), 1.57 (d, J=7.0 Hz, 3H). LCMS (m/z[M+H]⁺): 352.1. Chiral HPLC T_(R)=0.88 min.

Example 137:2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.30 (d, J=0.8 Hz, 1H), 8.75 (d, J=3.0 Hz,1H), 8.66 (d, J=5.9 Hz, 1H), 8.62 (dd, J=4.9, 0.8 Hz, 1H), 8.24 (dd,J=5.9, 0.9 Hz, 1H), 8.15 (dd, J=6.8, 4.9 Hz, 1H), 6.12-5.98 (m, 1H),3.51 (d, J=1.1 Hz, 3H), 1.57 (d, J=7.0 Hz, 3H). LCMS (m/z [M+H]⁺):352.1. Chiral HPLC TR=0.70 min.

Example 138:4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridin-2-amine

1H NMR (400 MHz, Acetone-d6) δ 9.15 (d, J=0.9 Hz, 1H), 8.55 (d, J=5.6Hz, 1H), 8.17-8.09 (m, 2H), 7.98 (dd, J=5.6, 1.0 Hz, 1H), 7.81-7.70 (m,2H), 1.61 (s, 3H), 1.00-0.94 (m, 2H), 0.91-0.84 (m, 2H). LCMS (m/z[M+H]⁺): 293.1.

Example 139:2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.20 (s, 1H), 8.74 (s, 1H), 8.68 (d,J=5.8 Hz, 1H), 8.63 (d, J=5.0 Hz, 1H), 8.33 (dd, J=5.8, 0.9 Hz, 1H),7.79 (d, J=4.9 Hz, 1H), 1.88 (d, J=1.0 Hz, 6H). LCMS (m/z [M+H]⁺):368.1.

Example 140:2,4-dimethyl-4-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol

1H NMR (400 MHz, Chloroform-d) δ 9.14 (s, 1H), 8.80 (s, 1H), 8.37 (t,J=5.8 Hz, 2H), 7.50-7.38 (m, 1H), 2.81 (s, 3H), 1.99 (s, 2H), 1.81 (s,6H), 1.49 (s, 6H). LCMS (m/z [M+H]⁺): 341.2.

Example 141:4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile

1H NMR (400 MHz, Acetone-d6) δ 9.23 (d, J=0.9 Hz, 1H), 9.09 (d, J=0.8Hz, 1H), 8.99 (d, J=5.2 Hz, 1H), 8.65 (d, J=5.6 Hz, 1H), 8.59-8.53 (m,1H), 8.42 (s, 1H), 8.07 (dd, J=5.7, 0.9 Hz, 1H), 1.62 (s, 3H), 1.04-0.97(m, 2H), 0.90-0.81 (m, 2H). LCMS (m/z [M+H]⁺): 303.1.

Example 142:2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared using procedures described in Example 114.

1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 9.06 (d, J=0.8 Hz, 1H), 8.98(dd, J=7.9, 1.7 Hz, 1H), 8.50 (t, J=5.5 Hz, 1H), 8.46 (d, J=5.5 Hz, 1H),8.19 (dd, J=4.7, 1.7 Hz, 1H), 8.06 (dd, J=5.6, 0.9 Hz, 1H), 7.16 (dd,J=7.9, 4.7 Hz, 1H), 3.62 (dt, J=8.1, 6.0 Hz, 2H), 2.96 (s, 3H),1.90-1.69 (m, 2H), 1.00 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 319.2.

Example 143:2-(1H-indazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J=0.9 Hz, 1H), 9.00 (dd, J=1.5, 0.8Hz, 1H), 8.85 (s, 1H), 8.61 (dd, J=8.9, 1.5 Hz, 1H), 8.52 (d, J=5.5 Hz,1H), 8.26 (s, 1H), 8.11 (dd, J=5.6, 0.9 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H),1.62 (s, 3H), 0.97-0.81 (m, 4H). LCMS (m/z [M+H]⁺): 317.1.

Example 144:2-(3,5-dimethyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, Methanol-d4) δ 9.06 (d, J=0.9 Hz, 1H), 8.50 (d, J=5.8Hz, 1H), 8.17 (dd, J=5.8, 0.9 Hz, 1H), 2.58 (s, 6H), 1.91 (d, J=1.1 Hz,6H). LCMS (m/z [M+H]⁺): 3511.

Example 145:N-(1,1,1-trifluoro-2-methylpropan-2-yl)-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (600 MHz, Methanol-d4) δ 9.06 (d, J=1.0 Hz, 1H), 8.54 (d, J=5.7Hz, 1H), 8.38-8.36 (m, 1H), 8.20 (dd, J=5.7, 0.9 Hz, 1H), 1.90 (d, J=1.1Hz, 6H). LCMS (m/z [M+H]⁺): 391.1.

Example 146:4-{4-[(4-hydroxy-2,4-dimethylpentan-2-yl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile

1H NMR (400 MHz, Chloroform-d) δ 9.55 (s, 1H), 9.38 (s, 1H), 9.07 (d,J=0.8 Hz, 1H), 8.95 (d, J=5.2 Hz, 1H), 8.62 (d, J=5.9 Hz, 1H), 8.27 (dd,J=5.2, 0.8 Hz, 1H), 7.86-7.80 (m, 1H), 2.02 (s, 2H), 1.84 (s, 6H), 1.53(s, 6H). LCMS (m/z [M+H]⁺): 363.2.

Example 147:2-(3,5-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared using procedures described in Example 120.

1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 9.14 (d, J=0.8 Hz, 1H), 8.71(s, 2H), 8.68 (d, J=5.6 Hz, 1H), 8.19 (dd, J=5.7, 1.0 Hz, 1H), 1.45 (s,3H), 0.87-0.77 (m, 2H), 0.75-0.64 (m, 2H). LCMS (m/z [M+H]⁺): 314.1.

Example 148:2-(2,3-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared using procedures described in Example 120.

1H NMR (400 MHz, Chloroform-d) δ 9.36 (s, 1H), 8.90 (dd, J=5.0, 0.9 Hz,1H), 8.85 (dd, J=1.6, 0.9 Hz, 1H), 8.69 (dd, J=5.1, 1.6 Hz, 1H), 8.66(d, J=5.7 Hz, 1H), 7.64-7.52 (m, 1H), 1.64 (s, 3H), 1.04-0.94 (m, 4H).LCMS (m/z [M+H]⁺): 314.1.

Example 149:N-(1-methylcyclopropyl)-2-(1,3-thiazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J=0.8 Hz, 1H), 9.08 (d, J=0.8 Hz,1H), 9.01 (s, 1H), 8.69-8.62 (m, 1H), 8.56 (d, J=5.5 Hz, 1H), 8.10 (dd,J=5.7, 0.9 Hz, 1H), 1.55 (s, 3H), 0.92-0.86 (m, 2H), 0.86-0.76 (m, 2H).LCMS (m/z [M+H]⁺): 284.1.

Example 150:N-(1-methylcyclopropyl)-2-[2-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Chloroform-d) δ 9.41 (s, 1H), 8.97-8.89 (m, 1H), 8.88(dd, J=1.5, 0.8 Hz, 1H), 8.70-8.66 (m, 1H), 8.66-8.61 (m, 1H), 7.89-7.77(m, 1H), 6.95-6.80 (m, 1H), 1.65 (s, 3H), 1.07-1.01 (m, 2H), 1.01-0.92(m, 2H). LCMS (m/z [M+H]⁺): 346.1.

Example 151:4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-2-carbonitrile

1H NMR (400 MHz, Chloroform-d) δ 9.36 (s, 1H), 8.90 (dd, J=5.0, 0.9 Hz,1H), 8.85 (dd, J=1.6, 0.9 Hz, 1H), 8.69 (dd, J=5.1, 1.6 Hz, 1H), 8.66(d, J=5.7 Hz, 1H), 7.64-7.52 (m, 1H), 1.64 (s, 3H), 1.04-0.94 (m, 4H).LCMS (m/z [M+H]⁺): 303.1.

Example 152:N-(1-methylcyclopropyl)-2-(1,2-oxazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Chloroform-d) δ 9.25 (s, 1H), 8.83 (s, 1H), 8.54 (dd,J=6.0, 0.7 Hz, 1H), 7.94-7.88 (br s, 1H), 7.32-7.27 (br s, 1H), 6.61(dd, J=6.0, 0.7 Hz, 1H), 0.91 (s, 3H), 0.85-0.73 (m, 4H). LCMS (m/z[M+H]⁺): 268.1.

Example 153:2-(dimethyl-1,2-oxazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.05 (d, J=0.8 Hz, 1H), 8.89 (s, 1H), 8.54(d, J=5.6 Hz, 1H), 8.10 (dd, J=5.7, 0.9 Hz, 1H), 2.90 (s, 3H), 2.65 (s,3H), 1.51 (s, 3H), 0.96-0.84 (m, 2H), 0.83-0.71 (m, 2H). LCMS (m/z[M+H]⁺): 296.1.

Example 154:N-(1-methylcyclopropyl)-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}pyrido[3,4-d]pyrimidin-4-amine

1H NMR (600 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.23 (d, J=0.8 Hz, 1H), 8.92(s, 1H), 8.59 (d, J=5.5 Hz, 1H), 8.38 (d, J=5.0 Hz, 1H), 8.22 (d, J=5.0Hz, 1H), 8.16 (dd, J=5.7, 0.9 Hz, 1H), 7.69 (dd, J=3.4, 2.0 Hz, 1H),7.61 (t, J=2.9 Hz, 1H), 1.61 (s, 3H), 0.99-0.94 (m, 2H), 0.92-0.87 (m,2H). LCMS (m/z [M+H]⁺): 317.1.

Example 155:N-propyl-2-{1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 9.07 (d, J=0.8 Hz, 1H), 8.96(dd, J=7.9, 1.6 Hz, 1H), 8.51 (dd, J=16.1, 5.5 Hz, 2H), 8.35-8.27 (m,2H), 8.09 (dd, J=5.7, 1.0 Hz, 1H), 7.25 (dd, J=7.9, 4.6 Hz, 1H), 3.67(dt, J=7.7, 5.9 Hz, 2H), 1.78 (h, J=7.4 Hz, 2H), 1.03 (t, J=7.4 Hz, 3H).LCMS (m/z [M+H]⁺): 305.1.

Example 156:N-propyl-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.19 (ddd, J=8.4, 1.5, 0.8 Hz, 1H), 9.13 (d,J=0.9 Hz, 1H), 9.02 (t, J=5.5 Hz, 1H), 8.65 (d, J=3.7 Hz, 1H), 8.55 (d,J=5.5 Hz, 1H), 8.48 (dd, J=4.6, 1.5 Hz, 1H), 8.16 (dd, J=5.5, 0.9 Hz,1H), 7.35 (dd, J=8.4, 4.6 Hz, 1H), 6.88 (dd, J=3.8, 0.8 Hz, 1H),3.71-3.63 (m, 2H), 1.85-1.74 (m, 2H), 1.04 (t, J=7.4 Hz, 3H). LCMS (m/z[M+H]⁺): 305.1.

Example 157:2-(3-methylpyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J=0.8 Hz, 1H), 8.73 (d, J=5.7 Hz,1H), 8.58 (d, J=5.8 Hz, 2H), 8.52 (dd, J=5.8, 1.0 Hz, 1H), 7.91 (s, 1H),7.75 (d, J=5.0 Hz, 1H), 2.59 (s, 3H), 1.85 (s, 6H). LCMS (m/z [M+H]⁺):348.1.

Example 158:N-(1-methylcyclobutyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.98 (s, 1H), 8.47 (d, J=5.5Hz, 1H), 8.38 (s, 1H), 8.27 (s, 1H), 8.14 (d, J=5.5 Hz, 1H), 8.07 (s,1H), 2.48-2.41 (m, 2H), 2.25 (td, J=9.0, 4.3 Hz, 2H), 1.99-1.81 (m, 2H),1.67 (s, 3H). LCMS (m/z [M+H]⁺): 281.1.

Example 159:N-(1-methylcyclopropyl)-2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared using procedures described in Example 120

1H NMR (400 MHz, Acetone-d6) δ 9.15 (d, J=0.9 Hz, 1H), 8.55 (d, J=5.6Hz, 1H), 8.17-8.09 (m, 2H), 7.98 (dd, J=5.6, 1.0 Hz, 1H), 7.81-7.70 (m,2H), 1.61 (s, 3H), 1.00-0.94 (m, 2H), 0.91-0.84 (m, 2H). LCMS (m/z[M+H]⁺): 279.1.

Example 160:4-{[2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol

1H NMR (500 MHz, DMSO-d6) δ 12.43 (s, 1H), 9.15 (s, 1H), 8.96 (s, 1H),8.46 (d, J=5.5 Hz, 1H), 7.69 (dd, J=5.7, 1.0 Hz, 1H), 5.61 (s, 1H), 2.61(s, 3H), 2.55 (s, 3H), 1.95 (s, 2H), 1.70 (s, 6H), 1.32 (s, 6H). LCMS(m/z [M+H]⁺): 355.2.

Example 161: 4N-propyl-2-{7H-pyrrolo[2,3-d]pyrimidin-5-yl}pyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J=2.5 Hz, 1H), 9.87 (s, 1H), 9.12(d, J=0.9 Hz, 1H), 8.86 (s, 1H), 8.60 (t, J=5.6 Hz, 1H), 8.53 (d, J=5.5Hz, 1H), 8.36 (d, J=2.6 Hz, 1H), 8.11 (dd, J=5.6, 0.9 Hz, 1H), 3.72-3.64(m, 2H), 1.85-1.72 (m, 2H), 1.03 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺):306.1.

Example 162:2-(3-chloropyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.15 (d, J=0.8 Hz, 1H), 8.89 (t, J=5.5 Hz,1H), 8.78 (d, J=0.6 Hz, 1H), 8.68 (dd, J=12.2, 5.2 Hz, 2H), 8.22 (dd,J=5.6, 0.9 Hz, 1H), 7.82 (dd, J=4.9, 0.6 Hz, 1H), 3.60-3.52 (m, 2H),1.76-1.65 (m, 2H), 0.95 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 300.1.

Example 163:2-(3-cyclopropyl-1H-pyrazol-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 8.98 (s, 1H), 8.54 (t, J=5.4Hz, 1H), 8.48 (d, J=5.5 Hz, 1H), 8.06 (dd, J=5.6, 0.9 Hz, 1H), 3.55 (dt,J=7.8, 5.9 Hz, 2H), 3.24 (t, J=7.1 Hz, 1H), 3.17 (d, J=5.1 Hz, 1H),1.77-1.63 (m, 2H), 0.93 (m, 7H). LCMS (m/z [M+H]⁺): 295.2.

Example 164:2-(3-methylpyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.14 (d, J=0.9 Hz, 1H), 8.79 (t, J=5.6 Hz,1H), 8.65 (d, J=5.5 Hz, 1H), 8.58-8.52 (m, 2H), 8.20 (dd, J=5.6, 1.0 Hz,1H), 7.85 (d, J=5.0 Hz, 1H), 3.62-3.54 (m, 2H), 2.60 (s, 3H), 1.77-1.66(m, 2H), 0.97 (t, J=7.4 Hz, 3H). LCMS (m/z [M+H]⁺): 280.2.

Example 165:2-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine

1H NMR (500 MHz, DMSO-d6) δ 9.06 (d, J=0.9 Hz, 1H), 8.97 (dd, J=7.9, 1.7Hz, 1H), 8.48 (d, J=5.5 Hz, 2H), 8.44 (s, 1H), 8.36 (dd, J=4.7, 1.7 Hz,1H), 8.09 (dd, J=5.7, 1.0 Hz, 1H), 7.29 (dd, J=7.9, 4.6 Hz, 1H), 3.94(s, 3H), 3.72-3.64 (m, 2H), 1.85-1.73 (m, 2H), 1.04 (t, J=7.4 Hz, 3H).LCMS (m/z [M+H]⁺): 319.2.

Example 166:2,4-dimethyl-4-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol

1H NMR (DMSO-d6) δ 1.29 (s, 6H), 1.72 (s, 6H), 1.98 (s, 2H), 5.5 (s, 1H,NH), 7.69 (d, J=3.6 Hz, 1H), 8.09 (s, 1H) 8.30 (s, 1H), 8.48 (d, J=3.6Hz, 1H), 8.99 (s, 1H), 9.13 (s, 1H). LCMS (m/z [M+H]⁺): 327.2.

Example 167:N-[(1R)-1-phenylethyl]-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (MeOH-d4) d 1.70 (d, J=6.8 Hz, 3H), 5.63 (m, 1H), 7.21 (m, 1H),7.32 (m, 2H), 7.49 (m, 2H), 8.17 (d, J=5.6 Hz, 1H), 8.20-8.23 (2H), 8.48(d, J=5.6 Hz, 1H), 9.00 (s, 1H). LCMS (m/z [M+H]⁺): 317.2.

Example 168:2-(5-methyl-1H-pyrazol-4-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (MeOH-d4) d1.70 (d, J=6.8 Hz, 3H), 2.54 (s, 3H), 5.65 (m, 1H),7.20 (m, 1H), 7.32 (m, 2H), 7.44 (m, 2H), 8.14-8.18 (2H), 8.46 (s, 1H),9.00 (s, 1H). LCMS (m/z [M+H]⁺): 331.2.

Example 169:N-methyl-2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (DMSO-d6) δ 0.95 (s, 4H), 1.70 (s, 3H), 3.38 (s, 3H), 4.35 (s,3H), 7.04 (d, J=2.0 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 8.16 (d, J=5.6 Hz,1H), 8.56 (d, J=5.6 Hz, 1H), 9.16 (s, 1H). LCMS (m/z [M+H]⁺): 295.2.

Example 170:2-(1-methyl-1H-pyrazol-5-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (MeOH-d4) d 1.60 (d, J=7.2 Hz, 3H), 4.02 (s, 3H), 5.46 (m, 1H),6.86 (d, J=2 Hz, 1H), 7.10 (m, 1H), 7.22 (m, 2H), 7.33 (m, 2H), 8.11 (d,J=5.6 Hz, 1H), 8.44 (d, J=5.6 Hz, 1H), 8.96 (s, 1H). LCMS (m/z [M+H]⁺):331.2.

Example 171:N-methyl-N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (DMSO-d6) δ 0.92 (s, 4H), 1.68 (s, 3H), 3.37 (s, 3H), 8.10 (d,J=5.6 Hz, 1H), 8.13 (s, 1H), 8.35 (s, 1H), 8.45 (m, 1H), 9.05 (s, 1H).LCMS (m/z [M+H]⁺): 281.1.

Example 172:2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.43 (d, J=5.7 Hz, 1H),7.87 (dd, J=5.7, 0.8 Hz, 1H), 7.48 (d, J=2.0 Hz, 1H), 7.08 (d, J=2.0 Hz,1H), 4.42 (s, 3H), 1.55 (s, 3H), 0.98-0.93 (m, 2H), 0.85-0.80 (m, 2H).LCMS (m/z [M+H]⁺): 281.1.

Example 173:2-(1-ethyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.10 (s, 1H), 8.52 (s, 1H), 7.98 (d,J=5.5 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.15 (d, J=2.0 Hz, 1H), 5.07 (q,J=7.1 Hz, 2H), 1.59 (s, 3H), 1.49 (t, J=7.1 Hz, 3H), 1.04-0.95 (m, 2H),0.93-0.76 (m, 2H). LCMS (m/z [M+H]⁺): 295.2.

Example 174:N-(1-methylcyclopropyl)-2-(pyridazin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 10.19 (dd, J=2.1, 1.3 Hz, 1H), 9.39 (dd,J=5.3, 1.2 Hz, 1H), 9.20 (d, J=0.8 Hz, 1H), 8.70 (dd, J=5.3, 2.2 Hz,1H), 8.57 (d, J=5.7 Hz, 1H), 8.01 (dd, J=5.7, 0.8 Hz, 1H), 1.64 (s, 3H),1.04-0.98 (m, 2H), 0.97-0.91 (m, 2H). LCMS (m/z [M+H]⁺): 279.1.

Example 175:N-(1-methylcyclopropyl)-2-(1,3-oxazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.12 (s, 1H), 8.53 (d, J=5.7 Hz, 1H),8.46 (s, 1H), 7.99-7.95 (m, 1H), 7.95 (s, 1H), 1.60 (s, 3H), 0.96 (m,2H), 0.88 (m, 2H). LCMS (m/z [M+H]⁺): 268.1.

Example 176:N-(1-methylcyclopropyl)-2-(1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine

N

1H NMR (400 MHz, Methanol-d4) δ 9.09 (d, J=0.8 Hz, 1H), 8.44 (s, 1H),7.94 (dd, J=5.7, 0.8 Hz, 1H), 7.69 (d, J=2.1 Hz, 1H), 7.11 (d, J=2.1 Hz,1H), 1.60 (s, 3H), 0.94 (m, 2H), 0.90-0.84 (m, 2H). LCMS (m/z [M+H]⁺):267.1.

Example 177:2-(1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.44 (d, J=5.7 Hz, 1H),8.16 (s, 1H), 7.98-7.90 (m, 2H), 1.60 (s, 3H), 0.95 (m, 2H), 0.92-0.84(m, 2H). LCMS (m/z [M+H]⁺): 267.1.

Example 178:2-(1-methyl-1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.41 (d, J=5.7 Hz, 1H),7.90 (dd, J=5.7, 0.8 Hz, 1H), 7.70 (d, J=0.8 Hz, 2H), 4.25 (s, 3H), 1.57(s, 3H), 0.99-0.94 (m, 2H), 0.85-0.79 (m, 2H). LCMS (m/z [M+H]⁺): 281.1.

Example 179:N-(1-methylcyclopropyl)-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.69 (d, J=8.3 Hz, 1H), 9.39 (s, 1H), 9.18(s, 1H), 8.88 (d, J=3.5 Hz, 1H), 8.72 (s, 1H), 8.58 (s, 1H), 8.18 (d,J=5.4 Hz, 1H), 7.71 (s, 1H), 7.10-7.02 (m, 1H), 1.59 (s, 3H), 0.99 (m,2H), 0.98-0.95 (m, 2H). LCMS (m/z [M+H]⁺): 317.1.

Example 180:N-(1-methylcyclopropyl)-2-(1H-1,2,3-triazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.64 (d, J=5.0 Hz, 1H), 8.51(s, 1H), 8.16 (d, J=5.5 Hz, 1H), 1.55 (s, 3H), 0.90 (m, 2H), 0.87 (m,2H). LCMS (m/z [M+H]⁺): 268.1.

Example 181:2-(3-methyl-1,2-oxazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Chloroform-d) δ 9.41 (s, 1H), 8.63 (d, J=5.6 Hz, 1H),7.43 (d, J=5.6 Hz, 1H), 6.98 (s, 1H), 2.44 (s, 3H), 1.60 (s, 3H),0.99-0.93 (m, 2H), 0.93-0.85 (m, 2H). LCMS (m/z [M+H]⁺): 282.1.

Example 182:N-(1-methylcyclopropyl)-2-(2H-1,2,3,4-tetrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 2H), 8.69 (s, 1H), 8.21 (d, J=5.2Hz, 1H), 1.57 (s, 3H), 0.89 (m, 2H), 0.85 (m, 2H). LCMS (m/z [M+H]⁺):269.1.

Example 183:2-(1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, Methanol-d4) δ 9.17 (s, 1H), 8.75 (d, J=5.6 Hz, 3H),8.37 (d, J=4.8 Hz, 1H), 8.32 (d, J=1.6 Hz, 2H), 8.14-8.09 (m, 2H), 5.89(q, J=7.3 Hz, 1H), 1.84 (d, J=7.2 Hz, 3H). LCMS (m/z [M+H]⁺): 318.1.

Example 184:N-tert-butyl-2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.13 (d, J=2.5 Hz, 1H), 8.61 (d, J=5.6 Hz,1H), 8.38 (t, J=5.2 Hz, 1H), 7.85 (d, J=7.9 Hz, 1H), 7.53 (d, J=1.9 Hz,1H), 6.97 (d, J=1.9 Hz, 1H), 4.37 (s, 3H), 1.59 (s, 9H). LCMS (m/z[M+H]⁺): 283.1.

Example 185:(1-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol

1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.62 (s, 1H), 8.38 (d, J=5.6Hz, 1H), 8.25-8.12 (m, 1H), 3.93 (s, 2H), 2.62 (s, 3H), 2.39 (t, J=7.8Hz, 4H), 1.85 (dq, J=12.0, 8.4 Hz, 2H). LCMS (m/z [M+H]⁺): 311.2.

Example 186:2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclobutyl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.78 (s, 1H), 8.62 (d, J=5.6Hz, 1H), 8.31-8.24 (m, 1H), 7.53-7.46 (m, 1H), 6.97 (d, J=1.9 Hz, 1H),4.38-4.31 (m, 3H), 2.48

-   -   2.41 (m, 2H), 2.22 (tt, J=8.4, 3.2 Hz, 2H), 1.98-1.78 (m, 2H),        1.65 (s, 3H). LCMS (m/z [M+H]⁺): 295.2.

Example 187:(1-{[2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol

1H NMR (400 MHz, DMSO-d6) δ 9.19-9.11 (m, 1H), 8.76 (s, 1H), 8.63 (d,J=5.7 Hz, 1H), 8.40 (dd, J=5.7, 0.7 Hz, 1H), 7.54-7.47 (m, 1H), 6.95 (d,J=1.9 Hz, 1H), 4.32 (s, 3H), 3.90 (s, 2H), 2.35 (t, J=7.2 Hz, 4H),1.92-1.76 (m, 2H). LCMS (m/z [M+H]⁺): 311.2.

Example 188:2-(1H-pyrazol-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 9.10 (s, 1H), 8.56 (d, J=5.6Hz, 1H), 8.27 (s, 2H), 8.16 (d, J=5.7 Hz, 1H), 1.61-1.53 (m, 2H), 1.33(d, J=6.0 Hz, 2H). LCMS (m/z [M+H]⁺): 321.1.

Example 189:2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 9.19 (d, J=0.7 Hz, 1H), 8.65(d, J=5.6 Hz, 1H), 8.23 (dd, J=5.7, 0.8 Hz, 1H), 7.58-7.47 (m, 1H), 7.07(d, J=1.9 Hz, 1H), 4.37 (s, 3H), 1.64-1.49 (m, 2H), 1.37 (d, J=5.8 Hz,2H). LCMS (m/z [M+H]⁺): 335.1.

Example 190:2-(3-methyl-1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.00 (d, J=0.7 Hz, 1H), 8.77 (s, 1H), 8.54(d, J=5.6 Hz, 1H), 8.51-8.47 (m, 2H), 8.30 (dd, J=5.6, 0.8 Hz, 1H), 7.99(s, 1H), 7.50-7.40 (m, 2H), 5.54 (d, J=6.6 Hz, 1H), 2.48 (s, 3H), 1.63(d, J=7.1 Hz, 3H). LCMS (m/z [M+H]⁺): 332.2.

Example 191:(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol

1H NMR (400 MHz, DMSO-d6) δ9.11 (m, 2H), 8.69 (m, 2H), 8.39 (s, 2H),3.92 (s, 2H), 2.39 (t, J=7.3 Hz, 4H), 1.87 (p, J=7.7, 6.6 Hz, 2H). LCMS(m/z [M+H]⁺): 297.1.

Example 192:(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)methanol

1H NMR (400 MHz, DMSO-d6) δ9.65 (s, 1H), 9.12 (s, 1H), 8.65 (d, J=5.4Hz, 1H), 8.41 (s, 2H), 8.26 (d, J=5.5 Hz, 1H), 3.73 (m, 2H), 1.06-0.96(m, 2H), 0.94-0.77 (m, 2H). LCMS (m/z [M+H]⁺): 283.1.

Example 193:2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.93 (d, J=6.4 Hz, 2H), 7.85(d, J=5.7 Hz, 1H), 7.51 (d, J=5.7 Hz, 1H), 7.31 (d, J=6.6 Hz, 2H), 6.63(d, J=2.0 Hz, 1H), 6.01 (d, J=2.0 Hz, 1H), 4.89 (q, J=7.2 Hz, 1H), 3.43(s, 3H), 1.00 (d, J=7.2 Hz, 3H). LCMS (m/z [M+H]⁺): 332.2.

Example 194:N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1HNMR (400 MHz, CD30D) d 9.03 (d, J=0.8 Hz, 1H), 8.43 (d, J=5.6 Hz, 1H),8.24 (d, J=0.8 Hz, 1H), 8.20 (s, 1H), 7.93 (dd, J=5.6, 0.8 Hz, 1H), 1.61(s, 3H), 0.99-0.95 (m, 2H), 0.90-0.85 (m, 2H). LCMS (m/z [M+H]⁺): 267.1.

Example 195:2-(1-ethyl-1H-pyrazol-4-yl)-N-(2-methylpropyl)pyrido[3,4-d]pyrimidin-4-amine

1HNMR (400 MHz, CDCl₃) δ 9.20 (s, 1H), 8.50 (d, J=5.6 Hz, 1H), 8.24 (s,1H), 8.20 (s, 1H), 7.43 (d, J=5.6 Hz, 1H), 5.87 (br s, 1H), 4.25 (q,J=7.2 Hz, 2H), 3.58 (dd, J=6.8, 6.0 Hz, 2H), 2.11 (nonet, J=6.8 Hz, 1H),1.56 (t, J=7.2 Hz, 3H), 1.06 (d, J=6.8 Hz, 6H). LCMS (m/z [M+H]⁺):297.2.

Example 196:2-(1-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine

1HNMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.72 (s, 1H), 8.46 (d, J=5.6Hz, 1H), 8.33 (s, 1H), 8.05 (s, 1H), 8.03 (d, J=5.6 Hz, 1H), 3.93 (s,3H), 1.54 (s, 3H), 0.90-0.80 (m, 4H). LCMS (m/z [M+H]⁺): 281.1.

Example 197:N-(1-amino-2-methylpropan-2-yl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.56 (d, J=5.4 Hz, 1H), 8.32(d, J=5.6 Hz, 1H), 8.28 (m, 1H), 7.76 (m, 3H), 3.63 (d, J=5.9 Hz, 2H),1.59 (s, 6H). LCMS (M/Z [M+H]⁺): 284.2.

Example 198:8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (intermediate 3c) as inScheme 3 using the procedure in Example 1 with 1-methylcyclopropanamine.1H NMR (400 MHz, Methanol-d4) δ 8.78-8.66 (m, 2H), 8.61-8.49 (m, 2H),8.30 (d, J=5.6 Hz, 1H), 7.94 (d, J=5.6 Hz, 1H), 1.63 (s, 3H), 1.04-0.96(m, 2H), 0.95-0.86 (m, 2H). LCMS (m/z [M+H]⁺): 312.1.

Example 199:8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared from Example 198 using the procedure forintermediate 6b with 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinaneinstead of 4-pyridine boronic acid. 1H NMR (500 MHz, DMSO-d6) δ 8.79 (d,J=5.1 Hz, 2H), 8.44 (d, J=5.6 Hz, 1H), 8.41 (d, J=5.0 Hz, 2H), 7.98 (dd,J=5.8, 0.8 Hz, 1H), 2.91 (s, 3H), 1.57 (s, 3H), 0.97-0.75 (m, 4H). LCMS(m/z [M+H]⁺): 292.2.

Example 251:N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared from4,5-dichloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (intermediate 5d)as in Scheme 5 using Step C of Example 1 with tert-butyl amine. 1H NMR(400 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.73 (s, 2H), 8.55 (s, 1H),8.46-8.39 (m, 2H), 1.72 (s, 9H). LCMS (M/Z [M+H]⁺): 314.1.

Example 252:5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

Title compound was prepared as Example 251 using4,5-dichloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (intermediate 5d)and 1-methylcyclopropan-1-amine. 1HNMR (400 MHz, CDCl₃) δ 9.18 (s, 1H),8.81 (br s, 2H), 8.50 (s, 1H), 8.42 (d, J=4.8 Hz, 2H), 8.00 (br s, 1H),1.64 (s, 3H), 1.00-0.90 (m, 4H). LCMS (M/Z [M+H]⁺): 312.1.

Example 253:5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine

N-(tert-butyl)-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine(Example 251, 10 mg, 0.032 mmol) and 2-(2-aminoethoxy)ethanol (670 mg,6.37 mmol) were dissolved in NMP (1 mL) in a 2 ml microwave reactor. Thereaction was heated at 160° C. for 1 hr (microwave irradiation). Thereaction was cooled to rt and was diluted with water (20 ml), extractedwith EtOAc (3×20 mL). The combined organic layers were dried overNa₂SO₄, filtered and evaporated. The residue was purified bymass-triggered HPLC to afford title compound (40%). 1H NMR (400 MHz,Methanol-d4) δ 8.63 (s, 1H), 8.23 (d, J=5.7 Hz, 2H), 8.18 (d, J=5.7 Hz,2H), 8.08 (s, 1H), 3.4-3.8 (m, 8H), 1.72 (s, 9H). LCMS (M/Z [M+H]⁺):383.2.

Example 254:N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

Title compound was prepared from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (intermediate 6b) using StepB as in Scheme 6.

Step B: In a 20 ml vial was added triethylamine (0.044 mL, 0.25 mmol),potassium fluoride (7.2 mg, 0.124 mmol),4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (intermediate 6b, 30 mg,0.124 mmol), and 4-methoxy-2-methylbutan-2-amine (16 mg, 0.137 mmol) inDMSO (1 mL) to give a yellow suspension. The reaction mixture wasstirred at 130° C. for 24 hrs. Solvent was evaporated under air flow.The residue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-10% MeOH/DCM to give the title compound (21%). 1H NMR(400 MHz, DMSO-d6) δ 9.20 (d, J=0.7 Hz, 1H), 8.71 (m, 2H), 8.48 (d,J=5.8 Hz, 1H), 8.09(ddd, J=12.9, 5.2, 1.3 Hz, 2H), 8.05 (m, 1H), 7.26(s, 1H), 6.81 (s, 1H), 3.50 (t, J=6.5 Hz, 2H), 3.22 (s, 3H), 2.11 (t,J=6.5 Hz, 2H), 1.52 (s, 6H). LCMS (M/Z [M+H]+): 323.2.

Examples 255-268

These compounds were synthesized according to the protocol described forExample 1 using 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine(intermediate 6b) and various amines respectively except speciallystated.

Example 255:N-[2-methyl-1-(propan-2-yloxy)propan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.18 (dd, J=7.4, 0.9 Hz, 1H), 8.80 (m, 2H),8.64 (d, J=5.6 Hz, 1H), 8.39 (dd, J=5.7, 0.9 Hz, 1H), 8.29 (d, J=6.1 Hz,2H), 7.70 (s, 1H), 3.90 (s, 2H), 3.55-3.50 (m, 1H), 1.61 (s, 6H), 1.00(s, 6H). LCMS (M/Z [M+H]⁺): 337.2.

Example 256:N-[(2S)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=7.4, 0.9 Hz, 1H), 8.73 (m, 2H),8.49 (m, 1H), 8.29 (d, J=6.1 Hz, 1H), 8.20 (m, 2H), 7.24 (s, 1H), 7.19(d, J=0.9 Hz, 1H), 4.02-3.99 (m, 1H), 1.79-1.75 (m, 1H), 1.69-1.65 (m,1H), 1.29 (m, 3H), 0.09 (t, J=4.9 Hz, 3H). LCMS (M/Z [M+H]⁺): 279.1.

Example 257:N-[(2R)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J=0.8 Hz, 1H), 8.73 (m, 2H), 8.49(d, J=5.8 Hz, 1H), 8.29 (dd, J=5.9, 0.9 Hz, 1H), 8.20 (m, 2H), 7.24 (s,1H), 7.19 (d, J=8.3 Hz, 1H), 4.02-3.99(dt, J=13.6, 6.5 Hz, 1H),1.79-1.75(dq, J=14.4, 7.2 Hz, 1H), 1.69-1.65 (m, 1H), 1.29 (d, J=6.4 Hz,3H), 0.09 (t, J=7.4 Hz, 3H). LCMS (M/Z [M+H]⁺): 279.3.

Example 258:N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.25 (dd, J=6.6, 0.8 Hz, 1H), 8.77 (m, 2H),8.75 (s, 1H), 8.24(dt, J=4.5, 1.9 Hz, 2H), 8.21 (s, 1H), 8.11 (m, 1H),7.52 (s, 1H), 3.62 (s, 2H), 3.34 (s, 3H), 1.52 (s, 6H). LCMS (M/Z[M+H]⁺): 309.4.

Example 259:N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.34 (dd, J=5.2, 0.9 Hz, 1H), 8.77(ddd,J=6.2, 4.4, 1.7 Hz, 2H), 8.50 (dd, J=6.5, 5.8 Hz, 1H), 8.20 (m, 2H),7.80 (s, 1H), 7.58 (s, 1H), 4.20-4.14 (m, 1H), 2.97 (s, 3H), 1.25 (s,6H). LCMS (M/Z [M+H]⁺): 279.4.

Example 260:3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butan-1-ol

1H NMR (400 MHz, DMSO-d6) δ 9.40 (d, J=0.9 Hz, 1H), 8.79 (m, 2H), 8.60(d, J=5.6 Hz, 1H), 8.26 (m, 2H), 7.95 (dd, J=5.6, 1.0 Hz, 1H), 7.89 (s,1H), 4.55 (t, J=6.8 Hz, 2H), 4.08-4.04(q, J=5.2 Hz, 1H), 1.95 (t, J=6.9Hz, 2H), 1.14 (s, 6H). LCMS (M/Z [M+H]⁺): 309.3.

Example 261: N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

Step 1: In a 20 mL microwave reactor was added PalladiumTetrakis (58.1mg, 0.050 mmol), potassium carbonate (1.256 mL, 2.51 mmol), and2,4-dichloro-1,7-naphthyridine (200 mg, 1.005 mmol) andpyridin-4-ylboronic acid (130 mg, 1.055 mmol) in Acetonitrile (Volume: 2mL) to give an orange suspension. The reaction mixture was stirred at120° C. for 60 min under microwave. The crude mixture was diluted withDCM, H₂O, separated and extracted with DCM×3. Combined the organiclayers and dried Na₂SO₄, filtered and concentrated. The residue waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-10% MeOH/DCM to give the product (62%). 1H NMR (400 MHz, DMSO-d6) δ9.58 (d, J=0.9 Hz, 1H), 8.85-8.78 (m, 4H), 8.32-8.29 (m, 2H), 8.11 (dd,J=5.8, 0.9 Hz, 1H). LCMS [M+H]=242.

Step 2: In a 40 ml vial was added potassium fluoride (11.54 mg, 0.199mmol), 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (40 mg, 0.166 mmol),and 2-methylpropan-2-amine (0.035 mL, 0.331 mmol) in DMSO (Volume: 2 mL)to give a yellow suspension. The reaction mixture was stirred at 130° C.for 24 hrs. Solvent was evaporated under air flow. The residue waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-10% MeOH/DCM to give the product (82%). 1H NMR (400 MHz, DMSO-d6) δ9.22 (d, J=0.7 Hz, 1H), 8.78-8.72 (m, 2H), 8.48 (d, J=5.8 Hz, 1H), 8.30(dd, J=6.0, 0.9 Hz, 1H), 8.15-8.06 (m, 2H), 7.28 (s, 1H), 6.73 (s, 1H),1.56 (s, 9H). LCMS [M+H]=279.2.

Example 262:2,2-dimethyl-1-[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]piperidin-4-ol

1H NMR (400 MHz, Acetone-d6) δ 9.41 (d, J=0.9 Hz, 1H), 8.80-8.75 (m,2H), 8.60 (d, J=5.7 Hz, 1H), 8.23-8.20 (m, 2H), 8.18-8.14 (m, 1H), 8.11(s, 1H), 4.15-3.99 (m, 1H), 3.52 (d, J=16.1 Hz, 1H), 3.16 (s, 1H),1.86-1.68 (m, 2H), 1.46 (d, J=16.0 Hz, 3H), 1.16-1.00 (m, 3H), 0.87 (d,J=6.8 Hz, 2H). LCMS (M/Z [M+H]⁺): 335.2.

Example 263:2,4-dimethyl-4-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}pentan-2-ol

1H NMR (400 MHz, Acetone-d6) δ 9.22 (d, J=0.8 Hz, 1H), 8.78-8.67 (m,2H), 8.63 (s, 1H), 8.40 (d, J=5.8 Hz, 1H), 8.19-8.09 (m, 2H), 7.81 (dd,J=5.9, 0.9 Hz, 1H), 7.36 (s, 1H), 2.08 (s, 2H), 1.75 (s, 6H), 1.47 (d,J=0.7 Hz, 6H). LCMS (M/Z [M+H]⁺): 337.2.

Example 264: N-cyclopentyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, Acetone-d6) δ 9.26 (d, J=0.9 Hz, 1H), 8.77-8.66 (m,2H), 8.44 (d, J=5.8 Hz, 1H), 8.23-8.15 (m, 2H), 8.06 (dd, J=5.8, 0.9 Hz,1H), 7.34 (s, 1H), 6.70 (d, J=6.6 Hz, 1H), 4.40-4.25 (m, 1H), 2.29-2.19(m, 2H), 1.86-1.68 (m, 6H). LCMS (M/Z [M+H]⁺): 279.1.

Example 265:dimethyl(3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butyl)amine

1H NMR (500 MHz, Methanol-d4) δ 9.40 (s, 1H), 8.91 (s, 2H), 8.62 (d,J=3.5 Hz, 1H), 8.48 (s, 2H), 8.40 (d, J=6.0 Hz, 1H), 7.46 (d, J=2.8 Hz,1H), 3.28-3.23 (m, 2H), 2.92

-   -   2.81 (m, 6H), 2.48 (dd, J=8.3, 5.1 Hz, 2H), 1.71 (d, J=2.7 Hz,        6H). LCMS (M/Z [M+H]⁺): 336.2.

Example 266: N,N-diethyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, Acetone-d6) δ 9.35 (d, J=0.9 Hz, 1H), 8.78-8.74 (m,2H), 8.51 (d, J=5.8 Hz, 1H), 8.23-8.19 (m, 2H), 7.90 (dd, J=5.8, 0.9 Hz,1H), 7.69 (s, 1H), 3.62 (q, J=7.1 Hz, 4H), 1.29 (t, J=7.1 Hz, 6H). LCMS(M/Z [M+H]+): 279.2.

Example 267:2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}propoxy)propan-2-ol

1H NMR (500 MHz, Methanol-d4) δ 9.25 (s, 1H), 8.73 (d, J=5.2 Hz, 2H),8.45 (d, J=5.9 Hz, 1H), 8.14-8.08 (m, 3H), 7.40 (s, 1H), 3.73 (s, 2H),3.39 (s, 2H), 1.63 (s, 6H), 1.18 (s, 6H). LCMS (M/Z [M+H]+): 367.2.

Example 268: N-propyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.22 (d, J=0.8 Hz, 1H), 8.77-8.70 (m, 2H),8.48 (d, J=5.8 Hz, 1H), 8.22-8.16 (m, 3H), 7.60 (t, J=5.5 Hz, 1H), 7.21(s, 1H), 3.43 (ddd, J=7.6, 6.6, 5.5 Hz, 2H), 1.79-1.67 (m, 2H), 1.01 (t,J=7.4 Hz, 3H). LCMS (M/Z [M+H]+): 265.1.

Example 269:N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine

Title compound was prepared from 2,4-dichloro-1,7-naphthyridine(intermediate 6a′) as in Scheme 6 using tert-butyl3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylateand tert-butyl amine. 1H NMR (400 MHz, DMSO-d6) 12.81 (s, 1H), 9.00 (d,J=0.7 Hz, 1H), 8.31 (d, J=5.8 Hz, 1H), 8.14 (dd, J=5.9, 0.8 Hz, 1H),8.00 (s, 1H), 6.97 (s, 1H), 6.36 (s, 1H), 2.65 (s, 3H), 1.51 (s, 9H).LCMS (M/Z [M+H]⁺): 282.4.

Example 270: N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine

Step 1: 2,4-dichloro-1,7-naphthyridine (6a, 100 mg, 0.502 mmol) wasstirred in dry DMF at room temperature. 4-(tributylstannyl)pyrimidine(165 uL, 0.502 mmol) was added then 41 mg of PdCl₂(dppf). CH₂Cl₂ adduct(41 mg, 0.05 mmol, orange solid) and finally Cul (10 mg, 0.05 mmol,beige solid). The reaction was stirred for 1 hour at 130° C. Thereaction was then concentrated and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% MeOH/DCM to afford the product4-chloro-2-(pyrimidin-4-yl)-1,7-naphthyridine (30%). LCMS (m/z [M+H]⁺):243.6.

Title compound: was then prepared with4-chloro-2-(pyrimidin-4-yl)-1,7-naphthyridine using the proceduredetailed in Step C, Example 1.

1H NMR (400 MHz, DMSO-d6) δ 9.48 (d, J=1.4 Hz, 1H), 9.25 (d, J=0.8 Hz,1H), 9.00 (d, J=5.3 Hz, 1H), 8.54 (m, 1H), 8.50 (m, 1H), 8.31 (dd,J=6.1, 0.9 Hz, 1H), 8.01 (s, 1H), 6.80 (s, 1H), 1.56 (s, 9H). LCMS (M/Z[M+H]⁺): 280.3.

Example 271:2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine

Step 1: 2,4-dichloro-1,7-naphthyridine(6a′, 400 mg, 2 mmol),Pd2(dba)₃(58 mg, 0.1 mmol) and PPh₃ (53 mg, 0.2 mmol) were stirred in 3mL of toluene at room temperature for 15 minutes. 680 microlitre oftributyl(1-ethoxyvinyl)stannane(680 microlitre, 2 mmol) in 1.5 mL oftoluene was then added and the reaction stirred at 110° C. for 1 hour.The reaction was cooled to room temperature. 4 mL of 1N HCl was addedand the mixture stirred overnight. The reaction was then neutralizedwith NaOH and extracted with ether. The crude residue was then purifiedby flash chromatography on a COMBIFLASH® system (ISCO) using 0-70%EtOAc/Hexane to afford the product1-(4-chloro-1,7-naphthyridin-2-yl)ethanone (40%). LCMS (m/z [M+H]⁺):207.5.

Step 2: 1-(4-chloro-1,7-naphthyridin-2-yl)ethanone (38 mg, 0.18 mmol)was stirred in DMF (2 mL) at room temperature and degassed with N₂.TEA(37 microlitre, 0.27 mmol) was added and stirred for 5 minutes then14 mg of KF(14 mg, 0.27 mmol). This mixture was stirred at roomtemperature for 15 minutes then 2-methylpropan-2-amine(28 microlitre,0.27 mmol) was added and degassed then stirred at 80° C. for two hrs.The reaction was then concentrated and purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-100% EtOAc/Hexane to afford theproduct 1-(4-(tert-butylamino)-1,7-naphthyridin-2-yl)ethanone (60%).LCMS (m/z [M+H]⁺): 244.3.

Step 3: 1-(4-(tert-butylamino)-1,7-naphthyridin-2-yl)ethanone(25 mg, 0.1mmol) was stirred in 0.8 mL of DMF/DMA at 110° C. for 6 hrs. Thereaction was then concentrated and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-100% EtOAc/Hexane to afford theproduct(E)-1-(4-(tert-butylamino)-1,7-naphthyridin-2-yl)-3-(dimethylamino)prop-2-en-1-one(30%). LCMS (m/z [M+H]⁺): 299.4.

Step 4:(E)-1-(4-(tert-butylamino)-1,7-naphthyridin-2-yl)-3-(dimethylamino)prop-2-en-1-one(8 mg, 0.027 mmol) was stirred in EtOH (0.7 mL) at room temperature.Guanidine nitrate (4 mg, 0.034 mmol) was added and the reaction stirredat 100° C. for 20 minutes. Sodium ethoxide (in EtOH, 20 microlitre,0.054 mmol) was then added and stirred at reflux overnight. Reaction wasthen concentrated and purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-20% MeOH/DCM to afford the title compound (30%).1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.48 (m, 1H), 8.41(m, 1H), 8.29 (dd, J=6.0, 0.9 Hz, 1H), 7.91 (s, 1H), 7.62 (d, J=5.0 Hz,1H), 6.74 (s, 2H), 6.65 (s, 1H), 1.56 (s, 9H). LCMS (M/Z [M+H]⁺): 295.4.

Examples 272-274

These compounds were synthesized according to the protocol used for thepreparation of Example 269 with 2,4-dichloro-1,7-naphthyridine (6a′) andvarious boronic acids or esters respectively.

Example 272:N-tert-butyl-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) 11.86 (s, 1H), 9.23 (d, J=0.8 Hz, 1H), 8.50(d, J=5.8 Hz, 1H), 8.39 (d, J=4.9 Hz, 1H), 8.30 (m, 1H), 7.63 (m, 1H),7.61 (m, 1H), 7.32 (s, 1H), 6.96 (dd, J=3.4, 1.9 Hz, 1H), 6.67 (s, 1H),1.56 (s, 9H). LCMS (M/Z [M+H]⁺): 318.4.

Example 273: N-tert-butyl-2-(pyridazin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.40 (d, J=0.8 Hz, 1H), 9.24(s, 1H), 8.50 (d, J=4.6 Hz, 1H), 8.36 (d, J=0.8 Hz, 1H), 8.31 (m, 1H),7.35 (s, 1H), 6.80 (s, 1H), 1.58 (s, 9H). LCMS (M/Z [M+H]⁺): 280.3.

Example 274:2-(2-aminopyridin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J=0.8 Hz, 1H), 8.45 (d, J=5.8 Hz,1H), 8.26 (m, 1H), 8.05(dd, J=5.3 0.7 Hz, 1H), 7.20 (m, 1H), 7.17 (m,1H), 7.14 (m, 1H), 6.63 (s, 1H), 6.09 (s, 2H), 1.55 (s, 9H). LCMS (M/Z[M+H]⁺): 294.4.

Examples 275-286

These compounds were synthesized according to the protocol used for thepreparation of Example 270 with 2,4-dichloro-1,7-naphthyridine(Intermediate 6a′, Scheme 6) and various organotin reagents and aminesrespectively.

Example 275:N,N-diethyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.33 (d, J=0.9 Hz, 1H), 8.78 (d, J=2.7 Hz,1H), 8.62 (dd, J=4.9, 1.1 Hz, 1H), 8.55 (d, J=5.9 Hz, 1H), 8.02 (dd,J=6.8, 4.9 Hz, 1H), 7.88 (dd, J=5.8, 0.9 Hz, 1H), 7.42 (d, J=1.5 Hz,1H), 3.50(q, J=7.0 Hz, 4H), 1.21 (t, J=7.0 Hz, 6H). LCMS (M/Z [M+H]⁺):297.1.

Example 276:(3-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-3-methylbutyl)dimethylamine

1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=0.8 Hz, 1H), 8.74 (d, J=2.8 Hz,1H), 8.60 (dd, J=4.9, 1.1 Hz, 1H), 8.53 (d, J=5.8 Hz, 1H), 8.04 (dd,J=6.9, 4.9 Hz, 1H), 7.91-7.83 (s, 1H), 7.25 (d, J=1.4 Hz, 1H), 2.60-2.54(m, 2H), 2.33-2.24 (m, 6H), 1.96-1.88 (m, 2H), 1.51 (s, 6H). LCMS (M/Z[M+H]⁺): 354.2.

Example 277:2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, DMSO-d6) δ 9.33 (m, 1H), 8.78 (d, J=2.7 Hz, 1H), 8.61(dd, J=4.9, 1.1 Hz, 1H), 8.55 (dd, J=5.9, 2.5 Hz, 1H), 8.05-8.01 (dd,J=6.8, 4.9 Hz, 1H), 7.85 (dd, J=5.8, 0.9 Hz, 1H), 7.41 (d, J=1.4 Hz,1H), 4.20-4.14 (m, 1H), 2.93 (s, 3H), 1.28 (m, 6H). LCMS (M/Z [M+H]⁺):297.1.

Example 278:2-(3-fluoropyridin-4-yl)-4-(piperidin-1-yl)-1,7-naphthyridine

1H NMR (400 MHz, DMSO-d6) δ 9.39 (d, J=0.8 Hz, 1H), 8.78 (d, J=2.7 Hz,1H), 8.63 (m, 1H), 8.60 (m, 1H), 8.03 (dd, J=6.8, 4.9 Hz, 1H), 7.85 (dd,J=5.8, 0.9 Hz, 1H), 7.50 (d, J=1.5 Hz, 1H), 3.32-3.28 (m, 4H), 1.87-1.79(m, 4H), 1.72-1.65 (m, 2H). LCMS (M/Z [M+H]+): 309.4.

Example 279:2-(3-fluoropyridin-4-yl)-4-(morpholin-4-yl)-1,7-naphthyridine

1H NMR (400 MHz, DMSO-d6) δ 9.40 (d, J=0.8 Hz, 1H), 8.79 (d, J=2.6 Hz,1H), 8.62 (m, 1H), 8.60 (m, 1H), 8.03 (dd, J=6.8, 4.9 Hz, 1H), 7.95 (dd,J=5.8, 0.9 Hz, 1H), 7.54 (d, J=1.4 Hz, 1H), 3.91-3.86 (m, 4H), 3.36-3.33(m, 4H). LCMS (M/Z [M+H]⁺): 311.1.

Example 280:N-tert-butyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine

Step 1: In a 20 mL microwave reactor 2,4-dichloro-1,7-naphthyridine (6a,100 mg, 0.502 mmol) was stirred in dry DMF (1 mL) at room temperature.3-fluoro-4-(tributylstannyl)pyridine (194 mg, 0.502 mmol) was added thenPdCl₂(dppf). CH₂Cl₂ adduct (41.0 mg, 0.050 mmol) and Cul (9.6 mg, 0.050mmol). The reaction was stirred for 0.5 hour at 130° C. The crudemixture was diluted with DCM, H₂O, separated and extracted with DCM×3.Combined the organic layers and dried Na₂SO₄, filtered and concentrated.The residue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-10% MeOH/DCM to give the product4-chloro-2-(3-fluoropyridin-4-yl)-1,7-naphthyridine (71%). 1H NMR (400MHz, Acetone-d6) δ 9.56 (d, J=0.9 Hz, 1H), 8.83 (d, J=5.8 Hz, 1H), 8.74(d, J=2.9 Hz, 1H), 8.67 (dd, J=5.0, 1.2 Hz, 1H), 8.44 (d, J=1.4 Hz, 1H),8.18 (dd, J=6.7, 4.9 Hz, 1H), 8.14 (dd, J=5.8, 0.9 Hz, 1H). LCMS[M+H]=260.

Step 2: In a 40 mL vial was added potassium fluoride (7 mg, 0.12 mmol),4-chloro-2-(3-fluoropyridin-4-yl)-1,7-naphthyridine (26 mg, 0.10 mmol),and 2-methylpropan-2-amine (0.035 mL, 0.331 mmol) in DMSO (Volume: 1 mL)to give a yellow suspension. The reaction mixture was stirred at 130° C.for 24 hrs. Solvent was evaporated under air flow. The residue waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-10% MeOH/DCM to give the product (42%). 1H NMR (400 MHz, Acetone-d6) δ9.24 (d, J=0.8 Hz, 1H), 8.65 (d, J=3.0 Hz, 1H), 8.59 (dd, J=4.9, 1.2 Hz,1H), 8.46 (d, J=5.9 Hz, 1H), 8.15 (dd, J=6.8, 4.9 Hz, 1H), 8.06 (dd,J=5.9, 0.9 Hz, 1H), 7.48 (d, J=1.4 Hz, 1H), 6.30 (s, 1H), 1.61 (s, 9H).LCMS (M/Z [M+H]⁺): 297.1.

Example 281:2-(3-fluoropyridin-4-yl)-N-(2-methylbutan-2-yl)-1,7-naphthyridin-4-amine

1H NMR (500 MHz, Acetone-d6) δ 9.24 (d, J=0.9 Hz, 1H), 8.64 (d, J=3.0Hz, 1H), 8.58 (dd, J=4.9, 1.2 Hz, 1H), 8.47 (d, J=5.9 Hz, 1H), 8.15 (dd,J=6.8, 4.9 Hz, 1H), 8.07 (dd, J=5.9, 0.9 Hz, 1H), 7.47 (d, J=1.4 Hz,1H), 6.12 (s, 1H), 2.03-1.98 (m, 2H), 1.56 (s, 6H), 0.94 (t, J=7.5 Hz,3H). LCMS (M/Z [M+H]+): 311.2.

Example 282:2-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-2-methylpropan-1-ol

1H NMR (400 MHz, Acetone-d6) δ 9.24 (d, J=0.8 Hz, 1H), 8.59 (dd, J=4.9,1.2 Hz, 1H), 8.49 (d, J=5.9 Hz, 1H), 8.15 (dd, J=6.8, 4.9 Hz, 1H), 7.97(dt, J=5.8, 1.3 Hz, 1H), 7.50 (d, J=1.4 Hz, 1H), 6.29 (s, 1H), 3.78 (d,J=5.6 Hz, 2H), 1.56 (s, 6H). LCMS (M/Z [M+H]+): 313.1.

Example 283:1-[2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-yl]-2,2-dimethylpiperidin-4-ol

1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.79 (d, 1H), 8.62 (d, 1H),8.60 (d, 1H), 8.03 (dd, 1H), 7.95 (d, 1H), 7.54 (s, 1H), 3.91-3.86 (m,4H), 3.36-3.33 (m, 4H). LCMS (M/Z [M+H]⁺): 311.3.

Example 284:2-(3-fluoropyridin-4-yl)-N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-1,7-naphthyridin-4-amine

1H NMR (500 MHz, Acetone-d6) δ 9.25 (d, J=0.9 Hz, 1H), 8.64 (d, J=3.0Hz, 1H), 8.59 (dd, J=4.9, 1.2 Hz, 1H), 8.52 (d, J=5.8 Hz, 1H), 8.15 (dd,J=6.8, 4.9 Hz, 1H), 7.89 (dd, J=5.9, 0.8 Hz, 1H), 7.54 (d, J=1.4 Hz,1H), 6.77 (s, 1H), 3.70-3.64 (m, 4H), 2.75 (s, 2H), 2.69-2.62 (m, 4H),1.60 (s, 6H). LCMS (M/Z [M+H]⁺): 382.2.

Example 285:N-tert-butyl-2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-amine

1H NMR (400 MHz, Acetone-d6) δ 9.20 (d, J=0.9 Hz, 1H), 8.78-8.69 (m,1H), 8.65 (d, J=4.9 Hz, 1H), 8.47 (d, J=5.9 Hz, 1H), 8.06 (dt, J=5.9,1.0 Hz, 1H), 7.79-7.70 (m, 1H), 7.25 (s, 1H), 1.60 (s, 9H). LCMS (M/Z[M+H]⁺): 313.1.

Example 286:2-(3-chloropyridin-4-yl)-N,N-diethyl-1,7-naphthyridin-4-amine

1H NMR (400 MHz, Acetone-d6) δ 9.31 (d, J=0.9 Hz, 1H), 8.75 (d, J=0.6Hz, 1H), 8.67 (d, J=4.9 Hz, 1H), 8.54 (d, J=5.9 Hz, 1H), 7.93 (dd,J=5.8, 0.9 Hz, 1H), 7.76 (dd, J=4.9, 0.6 Hz, 1H), 7.39 (s, 1H), 3.58 (q,J=7.1 Hz, 4H), 1.29 (t, J=7.1 Hz, 6H). LCMS (M/Z [M+H]⁺): 313.1.

Example 287:N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine

The title compound was synthesized according to the protocol describedfor Example 111 using 2,4-dichloropyrido[3,4-d]pyrimidine (intermediate2c) and propan-1-amine and tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxylate.1H NMR (500 MHz, DMSO-d6) δ 13.81 (s, 1H), 9.01 (d, J=0.9 Hz, 1H), 8.64(t, J=5.7 Hz, 1H), 8.61-8.57 (m, 1H), 8.55 (d, J=5.5 Hz, 1H), 8.11 (dd,J=5.6, 1.0 Hz, 1H), 3.64-3.56 (m, 2H), 1.73-1.62 (m, 2H), 0.96 (t, J=7.4Hz, 3H). LCMS (M/Z [M+H]⁺): 323.1.

Example 288:3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine

Step A: To a suspension of 3-methylisonicotinic acid (1) (10 g, 72.9mmol) in dichloromethane (200 mL) was added DMF (200 mL) at 0° C. Ethylchloroformate (8.6 mL, 12.5 g, 98.4 mmol) was added dropwise over 10 minat 0° C. to result in a mixture which was stirred at 0° C. for 15 min.tert-Butylamine (35.0 mL, 24.2 g, 330.0 mmol) was added dropwise over 15min to the reaction mixture at 0° C., and the reaction mixture wasconcentrated under reduced pressure to result in a residue which wassubjected to chromatography (hexanes:EtOAc) to give 2 (11.6 g, 83%). ¹HNMR (400 MHz, DMSO-d₆) δ 8.46 (s, 1H), 8.43 (d, J=5.0 Hz, 1H), 7.20 (d,J=5.0 Hz, 1H), 2.29 (s, 3H), 1.37 (s, 9H). LCMS (m/z [M+H]⁺): 193.

Step B: To a solution of 2 (11.6 g, 60.3 mmol) in THF (440 mL) was addeda solution of n-BuLi (58.0 mL, 145.0 mmol, 2.5 M in hexanes) dropwiseover 15 min at −45° C. under N₂ to result in a mixture which was stirredfor 45 min at −45° C. Then a solution of ethyl isonicotinate (10.0 g,66.3 mol) in THF (20 mL) was dropwise over 15 min at −45° C. Thereaction mixture was allowed to be warmed to room temperature over 2 h,and poured into a saturated aqueous NH₄Cl solution (1000 mL). Theorganic layer was separated, and the aqueous layer was extracted withEtOAc (3×400 mL). The combined organic layers were washed with saturatedaqueous NaCl solution (100 mL), dried over MgSO₄, and evaporated underreduced pressure to give crude 3. LCMS (m/z [M+H]⁺): 298. The crudeproduct was used in the next step without purification.

Step C: A solution of crude 3 of the Step B in acetic acid (200 mL) washeated at 100° C. for 16 h, cooled to room temperature and concentratedto −80 mL under reduced pressure. Water (120 mL) was added, and themixture was filtered to get a white precipitate which was washed withwater (2×50 mL), and dried under reduced pressure to afford crude 4(8.08 g). LCMS (m/z [M+H]⁺): 225. The crude product was used in the nextstep without purification.

Step D: To a suspension of crude 4 (8.08 g) in EtOH (100 mL) was addedan aqueous NH₄OH solution (80 mL, 28.5%) at room temperature to resultin a mixture which was stirred for 2.5 h, and evaporated to afford crude5. LCMS (m/z [M+H]⁺): 242. The crude product was used in the next stepwithout purification.

Step E: To a suspension of crude 5 of the Step D in EtOH (100 mL) wasadded water (20 mL) and then aqueous HCl solution (12 M, 25 mL) at 0° C.The reaction mixture was stirred for 19 h at room temperature, andfiltered to get white solid which was dried under reduced pressure toafford crude 6 (10.2 g). LCMS (m/z [M+H]⁺): 224. The crude product wasused in the next step without purification.

Step F: A suspension of crude 6 (10.2 g) in POCl₃ (75 mL) in an openChemGlass heavy wall round bottom pressure vessel (350 mL) was heatedvery slowly till the temperature reached 100° C., and stirred at 100° C.for 30 min. Then the pressure vessel was sealed, and the reactionmixture was heated at 135° C. for 14 h. The POCl₃ was removed underreduced pressure, and the residue was mixed with ice water (50 g of iceand 50 mL of water). The pH of the mixture was adjusted to˜7 withaqueous NaOH solution (1M), and then to ˜10 with saturated aqueousNa₂CO₃ solution. Filtration of the mixture gave a while solid which wasdried under reduce pressure to afford 7 (7.1 g, 49% yield for 5 stepsfrom 2). ¹H NMR (400 MHz, DMSO-d₆) δ 9.59 (s, 1H), 8.94 (s, 1H), 8.89(d, J=5.6 Hz, 1H), 8.78 (d, J=4.8 Hz, 2H), 8.15 (d, J=4.8 Hz, 2H), 8.14(d, J=5.6 Hz, 1H). LCMS (m/z [M+H]⁺): 242.

Step G: To a solution of intermediate 7 (60.0 mg, 0.25 mmol) and1-(trifluoromethyl)cyclopropan-1-amine (93.0 mg, 0.74 mmol) in dioxane(1.0 mL) was added bis(tri-tert-butylphosphine)palladium (13.0 mg, 0.2micromol) and sodium tert-butoxide (71.0 mg, 0.74 mmol). The reactionmixture was heated under N₂ at 130° C. for overnight, and concentratedresulting in a residue which was subjected to chromatography (MeOH/DCM)to afford the title compound. ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (s, 1H),8.95 (d, J=8.0 Hz, 2H), 8.75 (d, J=4.0 Hz, 1H), 8.71 (s, 1H), 8.57 (d,J=8.0 Hz, 2H), 8.34 (s, 1H), 8.31 d, J=4.0 Hz, 1H), 1.57 (m, 2H), 1.31(m, 2H). LCMS (m/z [M+H]⁺): 331.

Example 289: N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine

Step A: A suspension of 7-bromoisoquinoline (1) (300 mg, 1.44 mmol),pyridin-4-ylboronic acid (213 mg, 1.73 mmol),bis(triphenylphosphine)palladium(II) dichloride (PdCl₂(PPh₃)₂, 98 mg,0.14 mmol) and potassium carbonate (2 N, 4.3 ml) in DMF (7 mL) wasstirred and heated 100° C. for 3 hours. The reaction mixture wasfiltered through celite. The solution was diluted with water andextracted with EtOA×3. The combined organic layers were washed withsaturated aqueous NaCl solution, dried over MgSO₄, and evaporated underreduced pressure. The resulted crude was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCM toafford 7-(pyridin-4-yl)isoquinoline (2, 282 mg) as a pale brown oil.LCMS (m/z [M+H]⁺): 207.

Step B: To a solution of 7-(pyridin-4-yl)isoquinoline (2) (282 mg, 1.37mmol) in concentrated H₂SO₄ (3 mL) was added N-bromosuccinimide (NBS,488 mg, 2.74 mmol) at 0° C. The reaction mixture was allowed to bewarmed to room temperature over 2 h, and poured into ice-water (20 mL)and added saturated aqueous NaHCO₃ solution to pH ˜8. The aqueous layerwas extracted with EtOA×3. The combined organic layers were washed withsaturated aqueous NaCl solution, dried over MgSO₄, and evaporated underreduced pressure. The resulted crude was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCM toafford 5-bromo-7-(pyridin-4-yl)isoquinoline (3, 110 mg). LCMS (m/z[M+H]⁺): 285/287.

Step C: A suspension of 5-bromo-7-(pyridin-4-yl)isoquinoline (3) (30 mg,0.105 mmol), 1-methylcyclopropan-1-amine hydrochloride (28.3 mg, 0.263mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd₂dba₃, 9.7 mg, 0.0105mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP, 6.5 mg,0.0105 mmol) and sodium tert-butoxide (44.3 mg, 0.462) in toluene (1.5mL) was heated under N₂ at 90° C. for 16 h, cooled to room temperatureand concentrated. The residue was diluted in MeOH and filtered, thenpurified by mass-triggered preparative reverse phase HPLC with 10-90%acetonitrile/water to afford the title compound (6 mg). 1H NMR (400 MHz,Chloroform-d) δ 9.24 (s, 1H), 8.74 (s, 2H), 8.49 (s, 1H), 7.69-7.60 (m,2H), 7.59-7.56 (m, 1H), 7.52-7.46 (m, 1H), 7.39-7.36 (m, 1H), 1.52 (s,3H), 0.98-0.92 (m, 2H), 0.88-0.81 (m, 2H). LCMS (m/z [M+H]⁺): 276.

Example 290:2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine

The title compound was synthesized according to the protocol describedfor example 111 using 2,4-dichloropyrido[3,4-d]pyrimidine (intermediate2c) with tert-butyl 2-(trifluoromethyl)piperazine-1-carboxylate andpyridin-4-ylboronic acid followed by deprotection of the N-Boc groupwith TFA.

1H NMR (400 MHz, Chloroform-d) δ 9.46 (d, J=0.9 Hz, 1H), 8.83-8.78 (m,2H), 8.63 (d, J=5.8 Hz, 1H), 8.35-8.30 (m, 2H), 7.74 (dd, J=5.8, 0.9 Hz,1H), 5.80-5.67 (m, 1H), 4.07 (dt, J=24.3, 12.4 Hz, 2H), 3.65 (d, J=14.2Hz, 1H), 3.34 (d, J=14.6 Hz, 1H), 3.18 (d, J=13.5 Hz, 1H), 2.89 (t,J=11.0 Hz, 1H). LCMS (m/z [M+H]⁺): 361.1.

Example 291:4-(4-methylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine

The title compound was synthesized from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and1-methylpiperazine as follows: A solution of4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b, 30 mg,0.124 mmol), 1-methylpiperazine (50 mg, 0.499 mmol), potassium phosphate(132 mg, 0.621 mmol), RuPhos Pd G3 (16 mg, 0.019 mmol) and RuPhos (13mg, 0.028 mmol) in dioxane (2 mL) was heated under argon in a microwavevial at 130° C. for 10 min. The solvent was evaporated under reducedpressure. The residue was purified by flash chromatography using 20-100%ethyl acetate/cyclohexane, followed by 2-30% methanol/dichloromethane togive the title compound (21%).

1H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.76 (d, J=4.8 Hz, 2H), 8.55(d, J=5.7 Hz, 1H), 8.23 (d, J=4.8 Hz, 2H), 7.85 (d, J=5.7 Hz, 1H), 7.70(s, 1H), 3.37 (s, 4H), 2.63 (s, 4H), 2.30 (s, 3H). LCMS (m/z [M+H]⁺):306.3, Rt₁=0.38 min.

Example 292: 4-(piperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine

The title compound was synthesized in analogy of Example 291 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl piperazine-1-carboxylate (step 1) and Boc-deprotection usingHCl in diethylether (step 2).

Step 2 (Boc-Deprotection):

To a solution of tert-butyl4-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)piperazine-1-carboxylate (24mg, 0.061 mmol) in dichloromethane (5 mL) was added 6M HCl in diethylether (2 mL, 12 mmol). The resulting yellow suspension was stirred atr.t. for 1 h. The yellow precipitate was filtered off and washed withdichloromethane. The hydrochloride salt was dissolved in MeOH and thesolution was given on a PoraPak Rxn CX 20cc (2 g) cartridge. Thecartridge was then washed twice with 5 mL MeOH. Finally, the compoundwas eluted with 7N NH₃ in MeOH. The filtrate was evaporated underreduced pressure to give the title compound (76%).

1H NMR (400 MHz, DMSO-d₆) δ 9.37 (s, 1H), 8.76 (d, J=4.7 Hz, 2H), 8.55(d, J=5.8 Hz, 1H), 8.22 (d, J=4.8 Hz, 2H), 7.86 (d, J=5.7 Hz, 1H), 7.67(s, 1H), 3.28 (s, 4H), 2.99 (s, 4H). LCMS (m/z [M+H]⁺): 292.3, Rt₁=0.38min.

Example 293:4-(2-methylpiperidin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine

The title compound was synthesized in analogy of Example 291 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and2-methylpiperidine.

1H NMR (400 MHz, DMSO-d₆) δ 9.48-9.32 (m, 1H), 8.86-8.74 (m, 2H), 8.56(d, J=5.7 Hz, 1H), 8.30-8.16 (m, 2H), 7.86 (d, J=5.8 Hz, 1H), 7.72 (s,1H), 4.10 (d, J=6.3 Hz, 1H), 3.53-3.41 (m, 1H), 3.21 (d, J=12.4 Hz, 1H),2.05 (s, 1H), 1.86-1.74 (m, 3H), 1.62 (s, 2H), 1.06 (d, J=6.5 Hz, 3H).LCMS (m/z [M+H]⁺): 305.3, Rt₁=0.98 min.

Example 294:2-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclobutyl)-1,7-naphthyridin-4-amine

The title compound was synthesized from 4-coro-2-(pyndin-4-y)-1,-naphthyridine (Intermediate 6b) and1-(trifluoromethyl)cyclobutan-1-amine as follows: A solution of4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b, 100 mg,0.414 mmol), 1-(trifluoromethyl)cyclobutan-1-amine (115 mg, 0.828 mmol),potassium tert-butylate (139 mg, 1.241 mmol), Pd₂(dba)₃×CHCl₃ (43 mg,0.042 mmol) and PhCPhos (36 mg, 0.085 mmol) in dioxane (5 mL) was heatedunder argon in a microwave vial at 100° C. for 30 min. The solvent wasevaporated under reduced pressure. The residue was purified by flashchromatography using 20-100% ethyl acetate/cyclohexane, followed by2-10% methanol/dichloromethane to give the title compound (9%).

1H NMR (600 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.78-8.74 (m, 2H), 8.57 (d,J=5.8 Hz, 1H), 8.40 (d, J=5.8 Hz, 1H), 8.08-8.04 (m, 2H), 7.89 (s, 1H),6.94 (s, 1H), 2.85-2.78 (m, 2H), 2.74 (d, J=11.6 Hz, 2H), 2.04 (dd,J=18.6, 9.7 Hz, 2H). LCMS (m/z [M+H]⁺): 345.3, Rt₁=0.84 min.

Example 295:2-methyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine

The title compound was synthesized in analogy of Example 254 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl (1-amino-2-methylpropan-2-yl)carbamate (step 1) andBoc-deprotection using HCl in diethyl ether (step 2, described in

Example 292)

1H NMR (400 MHz, DMSO-d₆) δ 9.24 (s, 1H), 8.75 (d, J=5.14 Hz, 2H), 8.51(d, J=5.75 Hz, 1H), 8.26 (d, J=5.75 Hz, 1H), 8.21 (d, J=5.26 Hz, 2H),7.40 (s, 1H), 7.29 (br s, 1H), 3.38 (br s, 2H), 1.63-2.22 (m, 2H), 1.16(s, 6H). LCMS (m/z [M+H]⁺): 294.4, Rt₁=0.37 min.

Example 296: N-(oxetan-3-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

The title compound was synthesized in analogy of Example 254 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andoxetan-3-amine.

1H NMR (400 MHz, DMSO-d₆) δ 9.28 (s, 1H), 8.76 (d, J=5.99 Hz, 2H), 8.57(d, J=5.75 Hz, 1H), 8.29 (d, J=5.75 Hz, 1H), 8.21 (d, J=5.99 Hz, 2H),8.11 (br d, J=5.75 Hz, 1H), 7.03 (s, 1H), 4.99-5.17 (m, 3H), 4.73 (t,J=5.93 Hz, 2H). LCMS (m/z [M+H]⁺): 279.3, Rt₁=0.49 min.

Example 297:N-(1-methylcyclopropyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

The title compound was synthesized in analogy of Example 254 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and1-methylcyclopropan-1-amine.

1H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.78 (d, J=5.75 Hz, 2H), 8.48(d, J=5.87 Hz, 1H), 8.20 (d, J=5.87 Hz, 2H), 8.07 (d, J=5.87 Hz, 1H),7.69 (s, 1H), 3.29 (s, 3H), 3.14-3.20 (m, 1H), 0.89-0.97 (m, 2H),0.52-0.61 (m, 2H). LCMS (m/z [M+H]1): 277.3, Rt₁=0.73 min.

Example 298:4-(3,3-dimethylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine

The title compound was synthesized in analogy of Example 291 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl 2,2-dimethylpiperazine-1-carboxylate (step 1) andBoc-deprotection using HCl in diethylether (step 2, described in

Example 292)

1H NMR (400 MHz, DMSO-d₆) δ 9.41 (s, 1H), 8.79 (d, J=6.0 Hz, 2H), 8.59(d, J=5.7 Hz, 1H), 8.26 (d, J=6.0 Hz, 2H), 7.90 (d, J=5.8 Hz, 1H), 7.70(s, 1H), 3.31 (s, 1H), 3.25 (d, J=4.9 Hz, 2H), 3.10 (s, 4H), 1.26 (s,6H). LCMS (m/z [M+H]⁺): 320.3, Rt₁=0.45 min.

Example 299:2,2-dimethyl-4-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)morpholine

The title compound was synthesized in analogy of Example 291 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and2,2-dimethylmorpholine.

1H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.80 (d, J=5.9 Hz, 2H), 8.61(d, J=5.8 Hz, 1H), 8.27 (d, J=6.0 Hz, 2H), 7.93 (d, J=5.8 Hz, 1H), 7.75(s, 1H), 4.01-3.94 (m, 2H), 3.31 (s, 2H), 3.21 (s, 2H), 1.39 (s, 6H).LCMS (m/z [M+H]⁺): 321.3, Rt₁=0.81 min.

Example 300:N-(1-methylcyclobutyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

The title compound was synthesized in analogy of Example 254 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and1-methylcyclobutan-1-amine.

1H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.75 (d, J=5.9 Hz, 2H), 8.49(d, J=5.8 Hz, 1H), 8.26 (d, J=5.8 Hz, 1H), 8.08 (d, J=5.9 Hz, 2H), 7.64(s, 1H), 6.83 (s, 1H), 2.45 (t, J=10.4 Hz, 2H), 2.32 (t, J=9.2 Hz, 2H),1.97 (ddd, J=25.6, 20.0, 9.7 Hz, 2H), 1.62 (s, 3H). LCMS (m/z [M+H]⁺):291.3, Rt₁=0.70 min.

Example 301:2,2-dimethyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine

The title compound was synthesized in analogy of Example 254 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl (3-amino-2,2-dimethylpropyl)carbamate (step 1) andBoc-deprotection using HCl in diethyl ether (step 2, described inExample 292).

1H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.72 (d, J=5.9 Hz, 2H), 8.47(d, J=5.7 Hz, 1H), 8.17 (d, J=6.0 Hz, 2H), 8.01 (d, J=5.8 Hz, 1H), 7.31(s, 1H), 3.36 (s, 2H), 3.27 (s, 2H), 2.60 (s, 2H), 0.97 (s, 6H). LCMS(m/z [M+H]⁺): 308.3, Rt₁=0.41 min.

Example 302:N²,N²,2-trimethyl-N-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine

The title compound was synthesized in analogy of Example 254 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andN²,N²,2-trimethylpropane-1,2-diamine.

1H NMR (400 MHz, DMSO-d₆) δ 9.27 (s, 1H), 8.76 (d, J=5.9 Hz, 2H), 8.54(d, J=5.7 Hz, 1H), 8.23 (d, J=5.7 Hz, 2H), 8.05 (s, 1H), 7.33 (s, 1H),6.75 (s, 1H), 3.42 (s, 2H), 2.29 (d, J=15.6 Hz, 6H), 1.16 (s, 6H). LCMS(m/z [M+H]⁺): 322.3, Rt₁=0.39 min.

Example 303:4-(2-methylpiperazin-1-yi)-2-(pyridin-4-yi)-1,7-naphthyridine

The title compound was synthesized in analogy of Example 291 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl 3-methylpiperazine-1-carboxylate (step 1) andBoc-deprotection using HCl in diethyl ether (step 2, described in

Example 292)

1H NMR (400 MHz, DMSO-d₆) δ 9.44 (s, 1H), 8.80 (d, J=5.9 Hz, 2H), 8.60(d, J=5.7 Hz, 1H), 8.28 (d, J=5.9 Hz, 2H), 7.98 (d, J=5.7 Hz, 1H), 7.84(s, 1H), 4.08 (d, J=5.9 Hz, 1H), 3.60 (t, J=9.6 Hz, 1H), 3.42 (dd,J=12.3, 2.9 Hz, 1H), 3.29-3.17 (m, 2H), 3.12 (t, J=9.3 Hz, 1H), 2.98(dd, J=12.3, 4.1 Hz, 1H), 1.10 (d, J=6.5 Hz, 3H). LCMS (m/z [M+H]⁺):306.3, Rt₁=0.43 min.

Example 304:2-methyl-N¹-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine

The title compound was synthesized in analogy of Example 254 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andtert-butyl (3-amino-2-methylpropyl)carbamate (step 1) andBoc-deprotection using HCl in diethyl ether (step 2, described inExample 292).

1H NMR (400 MHz, DMSO-d₆) δ 9.25 (s, 1H), 8.76 (d, J=5.9 Hz, 2H), 8.51(d, J=5.7 Hz, 1H), 8.24-8.16 (m, 3H), 7.96 (s, 1H), 7.27 (s, 1H), 5.14(s, 2H), 3.50 (dd, J=13.4, 6.8 Hz, 1H), 3.40 (dd, J=13.8, 6.8 Hz, 1H),2.80 (dd, J=12.6, 6.0 Hz, 1H), 2.70 (dd, J=12.5, 6.4 Hz, 1H), 2.11 (dq,J=13.2, 6.6 Hz, 1H), 1.04 (d, J=6.7 Hz, 3H). LCMS (m/z [M+H]⁺): 294.3,Rt₁=0.37 min.

Example 305:(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)-1,7-naphthyridine

The title compound was synthesized in analogy of Example 291 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) and(R)-2-(trifluoromethyl)piperazine.

1H NMR (400 MHz, DMSO-d₆) δ 9.40 (s, 1H), 8.84-8.71 (m, 2H), 8.58 (d,J=5.7 Hz, 1H), 8.35-8.18 (m, 2H), 7.88 (d, J=5.8 Hz, 1H), 7.79 (s, 1H),3.79 (s, 1H), 3.66 (d, J=11.9 Hz, 1H), 3.53 (d, J=10.3 Hz, 1H),3.23-2.94 (m, 5H). LCMS (m/z [M+H]⁺): 360.3, Rt₁=0.71 min.

Example 306:N-(tert-butyl)-N-methyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine

The title compound was synthesized in analogy of Example 254 from4-chloro-2-(pyridin-4-yl)-1,7-naphthyridine (Intermediate 6b) andN,2-dimethylpropan-2-amine.

1H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.81 (d, J=5.6 Hz, 2H), 8.61(d, J=5.7 Hz, 1H), 8.24 (d, J=5.7 Hz, 2H), 8.13 (d, J=6.2 Hz, 2H), 2.97(s, 3H), 1.30 (s, 9H). LCMS (m/z [M+H]⁺): 293.3, Rt₁=1.01 min.

Example 307:N-(1-methylcyclobutyl)-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine

The title compound was synthesized in analogy of Example 270 in 2 stepsfrom 2,4-dichloro-1,7-naphthyridine (Intermediate 6a′) and4-(tributylstannyl)pyrimidine (step 1) and from4-chloro-2-(pyrimidin-4-yl)-1,7-naphthyridine and1-methylcyclobutan-1-amine (step 2).

1H NMR (400 MHz, DMSO-d₆) δ 9.37 (s, 1H), 9.27 (s, 1H), 9.01 (dd, J=5.2,1.5 Hz, 1H), 8.59-8.48 (m, 2H), 8.29 (d, J=5.9 Hz, 1H), 7.69 (s, 1H),7.58 (d, J=1.5 Hz, 1H), 2.46 (s, 2H), 2.27 (t, J=9.8 Hz, 2H), 1.98 (dq,J=31.9, 10.6, 10.2 Hz, 2H), 1.62 (s, 3H). LCMS (m/z [M+H]⁺): 292.4,Rt₁=0.82 min.

Example 308:N¹,N¹,3-trimethyl-N³-(2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized in analogy of Example 159 in 2 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1) and fromN3-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand 4-(tributylstannyl)pyrimidine (step 2).

¹H NMR (400 MHz, DMSO-d₆) b ppm 9.29-9.48 (m, 2H), 9.22 (s, 1H), 9.05(br d, J=5.01 Hz, 1H), 8.70 (br d, J=5.50 Hz, 1H), 8.38 (br d, J=5.01Hz, 1H), 7.91 (br d, J=4.89 Hz, 1H), 2.21-2.43 (m, 8H), 2.05 (br s, 2H),1.66 (s, 6H). LCMS (m/z [M+H]⁺): 338.3, Rt₁=0.47 min.

Example 309:N¹,N¹,3-trimethyl-N³-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized in analogy of Example 111 in 2 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1) and fromN3-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand tert-butyl3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate(CAS no. 1009071-34-4, step 2).

1H NMR (400 MHz, DMSO-d₆) b ppm 12.57-12.95 (m, 1H), 8.89-9.00 (m, 2H),8.48 (d, J=5.50 Hz, 1H), 7.93-8.29 (m, 1H), 7.75 (br d, J=5.38 Hz, 1H),2.55-2.84 (m, 5H), 2.25 (s, 6H), 1.99 (br s, 2H), 1.61 (s, 6H). LCMS(m/z [M+H]⁺): 340.3, Rt₁=0.48 min.

Example 310: tert-butyl(2-methyl-1-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-2-yl)carbamate

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andtert-butyl (1-amino-2-methylpropan-2-yl)carbamate.

1H NMR (400 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.78 (dd, J=8.9, 4.9 Hz, 3H),8.68 (d, J=5.5 Hz, 1H), 8.44-8.34 (m, 2H), 8.22 (d, J=5.7 Hz, 1H), 6.88(s, 1H), 3.90 (d, J=6.1 Hz, 2H), 1.35 (s, 6H), 1.27 (d, J=6.1 Hz, 9H).LCMS (m/z [M+H]⁺): 395.2, Rt₁=0.97 min.

Example 311: tert-butyl(2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)ethyl)carbamate

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andtert-butyl (2-aminoethyl)carbamate.

1H NMR (400 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.93-8.81 (m, 1H), 8.79-8.68(m, 2H), 8.64 (d, J=5.5 Hz, 1H), 8.39 (d, J=5.1 Hz, 2H), 8.13 (d, J=5.6Hz, 1H), 7.03 (t, J=6.0 Hz, 1H), 3.73 (q, J=6.2 Hz, 2H), 3.40-3.32 (m,2H), 1.33 (s, 9H). LCMS (m/z [M+H]⁺): 367.2, Rt₁=0.78 min.

Example 312:2-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine

The title compound was synthesized in analogy of Example 1 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c)and tert-butyl (1-amino-2-methylpropan-2-yl)carbamate (step 1) andBoc-deprotection using TFA (step 2).

1H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.83-8.74 (m, 2H), 8.65 (d,J=5.5 Hz, 1H), 8.38-8.33 (m, 2H), 8.31 (d, J=5.6 Hz, 1H), 3.70 (s, 2H),1.13 (s, 6H). LCMS (m/z [M+H]⁺): 295.2, Rt₁=0.41 min.

Example 313:N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)ethane-1,2-diamine

The title compound was synthesized in analogy of Example 1 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c)and tert-butyl (2-aminoethyl)carbamate (step 1) and Boc-deprotectionusing TFA (step 2).

1H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.81-8.71 (m, 2H), 8.64 (d,J=5.5 Hz, 1H), 8.43-8.30 (m, 2H), 8.20 (d, J=5.6 Hz, 1H), 3.73 (t, J=6.4Hz, 2H), 2.94 (t, J=6.4 Hz, 2H). LCMS (m/z [M+H]⁺): 267.2, Rt₁=0.38 min.

Example 314:N,N,2-trimethyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and2-amino-N,N,2-trimethylpropanamide.

1H NMR (400 MHz, DMSO-d₆) δ 9.22 (s, 1H), 8.74 (dd, J=6.9, 2.4 Hz, 3H),8.68 (d, J=5.6 Hz, 1H), 8.38 (d, J=5.6 Hz, 1H), 8.36-8.31 (m, 2H),2.95-2.69 (m, 6H), 1.6(s, 6H). LCMS (m/z [M+H]⁺): 337.2, Rt₁=0.58 min.

Example 315:N¹,3-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andN¹,3-dimethylbutane-1,3-diamine.

1H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.83-8.69 (m, 2H), 8.55 (d,J=5.8 Hz, 1H), 8.40-8.29 (m, 2H), 8.12 (d, J=5.9 Hz, 1H), 4.04-3.92 (m,2H), 3.52 (s, 3H), 1.87-1.74 (m, 2H), 1.56 (s, 2H), 1.15 (s, 6H). LCMS(m/z [M+H]⁺): 323.2, Rt₁=0.49 min.

Example 316: tert-butyl(2,2-dimethyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)carbamate

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andtert-butyl (3-amino-2,2-dimethylpropyl)carbamate.

1H NMR (600 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.77 (d, J=4.9 Hz, 2H), 8.68(d, J=5.4 Hz, 1H), 8.61 (t, J=6.4 Hz, 1H), 8.43-8.30 (m, 2H), 8.17 (d,J=5.5 Hz, 1H), 7.01 (t, J=6.6 Hz, 1H), 3.62 (d, J=6.2 Hz, 2H), 2.93 (d,J=6.3 Hz, 2H), 1.38 (s, 9H), 0.92 (s, 6H). LCMS (m/z [M+H]⁺): 409.3,Rt₁=1.06 min.

Example 317:2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine

The title compound was synthesized in analogy of Example 1 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c)and tert-butyl (3-amino-2,2-dimethylpropyl)carbamate (step 1) andBoc-deprotection using TFA (step 2).

1H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.81-8.71 (m, 2H), 8.65 (d,J=5.6 Hz, 1H), 8.39-8.29 (m, 2H), 8.16 (d, J=5.6 Hz, 1H), 3.67 (s, 2H),0.95 (s, 6H). LCMS (m/z [M+H]⁺): 309.2, Rt₁=0.43 min.

Example 318:3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butanamide

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and3-amino-3-methylbutanamide.

1H NMR (400 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.94-8.80 (m, 2H), 8.77-8.60(m, 2H), 8.48-8.35 (m, 2H), 8.10 (d, J=5.6 Hz, 1H), 7.59 (s, 1H), 7.12(s, 1H), 2.76 (s, 2H), 1.71 (s, 6H). LCMS (m/z [M+H]⁺): 323.3, Rt₁=0.57min.

Example 319:(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and(R)-2-(trifluoromethyl)piperazine.

1H NMR (600 MHz, DMSO-d₆) δ 9.31 (s, 1H), 8.79 (d, J=5.3 Hz, 2H), 8.63(d, J=5.6 Hz, 1H), 8.31 (d, J=4.9 Hz, 2H), 7.97 (d, J=5.7 Hz, 1H),4.60-4.49 (m, 1H), 4.28-4.21 (m, 1H), 3.82-3.74 (m, 1H), 3.71-3.64 (m,1H), 3.63-3.56 (m, 1H), 3.20-3.12 (m, 1H), 3.12-3.04 (m, 1H), 2.96-2.87(m, 1H). LCMS (m/z [M+H]⁺): 361.1, Rt₁=0.72 min.

Example 320:2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and2,3-dimethylbutane-2,3-diamine.

1H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.88-8.74 (m, 2H), 8.66 (d,J=5.5 Hz, 1H), 8.40-8.19 (m, 2H), 7.74 (d, J=5.5 Hz, 1H), 1.63 (s, 6H),1.21 (s, 6H). LCMS (m/z [M+H]⁺): 323.2, Rt₁=0.48 min.

Example 321:(S)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and(S)-2-(trifluoromethyl)piperazine.

1H NMR (600 MHz, DMSO-d₆) δ 9.31 (s, 1H), 8.81-8.77 (m, 2H), 8.63 (d,J=5.7 Hz, 1H), 8.34-8.29 (m, 2H), 7.97 (d, J=5.6 Hz, 1H), 4.57-4.52 (m,1H), 4.28-4.21 (m, 1H), 3.83-3.74 (m, 1H), 3.71-3.65 (m, 1H), 3.63-3.57(m, 1H), 3.20-3.14 (m, 1H), 3.11-3.05 (m, 1H), 2.95-2.88 (m, 1H). LCMS(m/z [M+H]⁺): 361.1, Rt₁=0.72 min.

Example 322: ethyl2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andethyl 2-amino-2-methylpropanoate.

1H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.83 (s, 1H), 8.80-8.74 (m,2H), 8.69 (d, J=5.6 Hz, 1H), 8.39 (d, J=5.6 Hz, 1H), 8.28-8.23 (m, 2H),3.99 (q, J=7.1 Hz, 2H), 1.69 (s, 6H), 0.91 (t, J=7.1 Hz, 3H). LCMS (m/z[M+H]⁺): 338.2, Rt₁=0.78 min.

Example 323:N¹,N¹,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andN¹,N¹,2,2-tetramethylpropane-1,3-diamine.

1H NMR (600 MHz, DMSO-d₆) δ 9.20-9.17 (m, 1H), 9.04 (t, J=5.6 Hz, 1H),8.80-8.74 (m, 2H), 8.68-8.63 (m, 1H), 8.38-8.31 (m, 2H), 8.13-8.08 (m,1H), 3.72-3.65 (m, 2H), 2.36-2.28 (m, 8H), 0.99 (s, 6H). LCMS (m/z[M+H]⁺): 337.2, Rt₁=0.46 min.

Example 324:4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine

The title compound was synthesized according to the scheme below from3-aminoisonicotinamide and 4-amino-1,2,5-oxadiazole-3-carboxylic acid.

Step 1:4-amino-N-(4-carbamoylpyridin-3-yl)-1,2,5-oxadiazole-3-carboxamide In atwo necked R. B. flask fitted with thermometer pocket and nitrogeninlet, TBTU (4.75 g, 14.8 mmol) and N, N-diisopropylethylamine (2.6 mL,14.8 mmol) were added to a stirred solution of 3-aminoisonicotinamide(1.7 g, 12.4 mmol) and 4-amino-1,2,5-oxadiazole-3-carboxylic acid (1.92g, 14.8 mmol) in DMF (40 mL) at 25-30° C. Resulting reaction mixture wasstirred for 72 h at 25-30° C. Reaction mixture was poured over water(200 mL) and precipitated solid was collected by filtration. Washed withwater (100 mL). Purification was done by trituration by using 20% ethylacetate in n-Hexane to get4-amino-N-(4-carbamoylpyridin-3-yl)-1,2,5-oxadiazole-3-carboxamide (0.84g, 27.45%) as Off-white solid.

¹H-NMR (400 MHz, DMSO-d₆): δ 12.60 (s, 1H, Exchangeable with D₂O), 9.70(s, 1H), 8.64 (s, 1H, Exchangeable with D₂O), 8.52 (d, J=4.4 Hz, 1H),8.81 (s, 1H, Exchangeable with D₂O), 7.80 (d, J=4.8 Hz, 1H), 6.25 (s,2H, Exchangeable with D₂O). LCMS (m/z [M+H]⁺): 249.1, Rt₂=1.312 min.

Step 2: 2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-one

In a sealed tube, DBU (0.98 g, 6.45 mmol) was added to a stirredsuspension of4-amino-N-(4-carbamoylpyridin-3-yl)-1,2,5-oxadiazole-3-carboxamide (0.8g, 3.22 mmol) in tert-butanol (8.0 mL). Sealed the cap and heatedreaction mixture to 100° C. for 16 h. Solvent was evaporated, resultingresidue was diluted with water (20 mL) and stirred for 0.5 h at 25-30°C. Precipitated solid was collected by filtration, washed solid withwater (2×10 mL) and n-hexane (10 mL). The obtained solid were dried at a55° C. in rotating flask to obtain (0.421 g, 56.73%) of2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-one asoff-white solid.

¹H-NMR (400 MHz, DMSO-d₆): δ 13.31 (s, 1H, Exchangeable with D₂O), 9.36(s, 1H), 8.74 (d, J=5.2 Hz, 1H), 8.01 (d, J=5.2 Hz, 1H), 6.95 (s, 2H,Exchangeable with D₂O). LCMS (m/z [M+H]⁺): 231.2, Rt₂=1.279 min.

Step 3: 2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4-yl4-methylbenzenesulfonate In a two necked R. B. flask fitted with,thermometer pocket and nitrogen inlet, DMAP (0.015 g, 0.116 mmol) wasadded to a stirred solution of2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (0.27g, 1.168 mmol) and triethylamine (0.245 mL, 1.752 mmol) indichloromethane (10 mL) at 0° C. p-TsCI (0.245 g, 1.168 mmol) was addedand stirred resulting reaction mixture for 0.5 h. Allowed reactionmixture to warm to 25-30° C. by removing ice bath and stirred at 25-30°C. for 1.5 h. The mixture was diluted with DCM (20 mL), washed withwater (2×20 mL) and brine (20 mL). Dried organic layers over anhydroussodium sulphate and filtered. Filtrate was concentrated on rotavapor toget 2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4-yl4-methylbenzenesulfonate (0.285 g, 63.21%) as crude. Crude was used fornext step without purification. LCMS (m/z [M+H]⁺): 385.1, Rt₂=1.779 min.

Step 4:4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amineIn a two necked R. B. flask fitted with, thermometer pocket and nitrogeninlet, tert-Butyl amine (0.0714 g, 0.976 mmol) was added to a stirredsolution of the2-(4-amino-1,2,5-oxadiazol-3-yl)pyrido[3,4-d]pyrimidin-4-yl4-methylbenzenesulfonate (0.25 g, 0.651 mmol) in DMSO (2.5 mL) followedby added KF (0.0566 g, 0.976 mmol) and triethylamine (0.18 mL, 1.304mmol) at 25-30° C. After being stirred at rt for 45 min reaction mixturewas poured over water (25 mL). Precipitated solid was collected byfiltration. Washed solid with water (25 mL) and n-hexane (10 mL).Purification was done by trituration in n-hexane: diethyl ether; 1:1(5mL) to get4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine(0.135 g, 72.75%) as Off-white solid.

¹H NMR (400 MHz, DMSO-d₆) b ppm 9.30 (s, 1H), 8.67 (d, J=5.57 Hz, 1H),8.39 (d, J=5.58 Hz, 1H), 8.00 (s, 1H), 6.89 (s, 2H), 1.60 (s, 9H). LCMS(m/z [M+H]⁺): 286.1, Rt₁=0.92 min.

Example 325:N²,N²,2-trimethyl-Ni-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andN²,N²,2-trimethylpropane-1,2-diamine.

1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.85-8.72 (m, 2H), 8.65 (d,J=5.6 Hz, 1H), 8.36-8.31 (m, 2H), 8.27 (t, J=6.4 Hz, 2H), 3.79 (d, J=5.6Hz, 2H), 2.27 (s, 6H), 1.09 (s, 6H). LCMS (m/z [M+H]⁺): 323.2, Rt₁=0.44min.

Example 326:2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) and2-amino-2-methylpropanamide.

1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.77-8.71 (m, 2H), 8.67 (d,J=5.5 Hz, 1H), 8.49 (s, 1H), 8.39 (d, J=5.7 Hz, 1H), 8.36-8.30 (m, 2H),7.29 (s, 1H), 6.84 (s, 1H), 1.63 (s, 6H). LCMS (m/z [M+H]⁺): 309.2,Rt₁=0.50 min.

Example 327:(S)-1,1,1-trifluoro-2-methyl-3-((2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)amino)propan-2-ol

The title compound was synthesized according to the scheme below from2,4-dibromo-1,7-naphthyridine.

Step 1: A solution of 2,4-dibromo-1,7-naphthyridine (350 mg, 1.22 mmol),4-pyridinylboronic acid (194 mg, 1.58 mmol), potassium phosphate (1032mg, 4.86 mmol) and Pd(PPh₃)₄(140 mg, 0.122 mmol) in dioxane (6 mL)/water(1.5 mL) was heated under argon in a microwave vial at 110° C. for 60min. Water was added at r.t. and the reaction mixture was extracted 3times with dichloromethane. The combined organic phases were washed withbrine, dried with sodium sulfate and the solvent was evaporated underreduced pressure. The residue was purified by flash chromatography using0-100% ethyl acetate/cyclohexane to give4-bromo-2-(pyridin-4-yl)-1,7-naphthyridine (44%). LCMS (m/z [M+H]⁺):286.0/288.0, Rt₁=0.83 min.

Step 2: The title compound was synthesized in analogy of Example 254from 4-bromo-2-(pyridin-4-yl)-1,7-naphthyridine and(S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol [1334547-38-4].

¹H NMR (600 MHz, DMSO-d6) δ ppm 9.26 (s, 1H), 8.77 (d, J=5.69 Hz, 2H),8.53 (d, J=5.87 Hz, 1H), 8.20 (d, J=5.69 Hz, 1H), 8.14-8.18 (m, 2H),7.48-7.55 (m, 2H), 6.34 (s, 1H), 3.82 (dd, J=14.58, 6.33 Hz, 1H),3.69-3.76 (m, 1H), 1.40 (s, 3H). LCMS (m/z [M+H]⁺): 349.3, Rt=0.62 min.

Example 328: tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andtert-butyl (3-amino-3-methylbutyl)carbamate.

1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.88-8.73 (m, 2H), 8.64 (d,J=5.6 Hz, 1H), 8.37 (d, J=5.7 Hz, 1H), 8.33-8.25 (m, 2H), 7.73 (s, 1H),6.75 (s, 1H), 2.97 (t, J=7.6 Hz, 2H), 2.31-2.19 (m, 2H), 1.60 (s, 6H),1.25 (s, 9H). LCMS (m/z [M+H]⁺): 409.2, Rt₁=0.96 min.

Example 329:N¹,N¹,N³,2,2-pentamethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine

The title compound was synthesized in analogy of Example 1 from4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c) andN¹,N¹,N3,2,2-pentamethylpropane-1,3-diamine.

1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.76 (d, 2H), 8.54 (d, J=5.9Hz, 1H), 8.34 (d, 2H), 8.23 (d, J=5.9 Hz, 1H), 4.11 (s, 2H), 3.66 (s,3H), 2.30 (s, 6H), 2.23 (s, 2H), 0.96 (s, 6H). LCMS (m/z [M+H]⁺): 351.2,Rt₁=0.50 min.

Example 330:N¹,N-diethyl-3-methyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized according to the scheme below fromExample 328 (tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate).

Step 1: To a solution of tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate(80 mg, 0.196 mmol) in dichloromethane (2 mL) was added TFA (0.377 mL,4.90 mmol) and the resulting yellow solution was stirred at r.t. for 2h. Then the solution was evaporated to dryness. The residue wasdissolved in MeOH and the solution was given on a PoraPak Rxn CX 20cc (2g) cartridge. The cartridge was then washed twice with 5 mL MeOH.Finally, the compound was eluted with 7N NH₃ in MeOH. The filtrate wasevaporated under reduced pressure to give3-methyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine(66%). LCMS (m/z [M+H]⁺): 309.1, Rt₁=0.53 min.

Step 2: A solution of3-methyl-N3-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine(40 mg, 0.130 mmol), acetic acid (0.037 mL, 0.649 mmol) and acetaldehyde(57 mg, 1.297 mmol) in dichloromethane (5 mL) was stirred at r.t. for 1h. Then NaBH(OAc)₃ (137 mg, 0.649 mmol) was added and the mixture wasstirred for further 1 h at r.t. Saturated NaHCO₃-solution was added andthe aqueous phase was extracted 3 times with dichloromethane (3×20 mL).The combined organic phases were washed with brine, dried with sodiumsulfate and the solvent was evaporated under reduced pressure. Theresidue was purified by flash chromatography using a gradient of 12-100%ethyl acetate/cyclohexane followed by a gradient of 2-30% methanol (+10%7N NH₃ in methanol)/dichloromethane to give a yellow oil. The oil wastaken up in dichloromethane (2 mL) and 4N HCL in dioxane (0.2 mL) wasadded. The precipitate was filtered off and washed with pentane to givethe title compound as HCL salt (17%).

1H NMR (400 MHz, Methanol-d4) δ 9.52 (s, 1H), 9.22-9.02 (m, 4H), 8.82(d, J=6.2 Hz, 1H), 8.74 (dd, J=6.2, 0.9 Hz, 1H), 3.27 (dd, J=8.4, 4.7Hz, 2H), 3.21 (q, J=7.3 Hz, 4H), 2.84-2.70 (m, 2H), 1.79 (s, 6H), 1.23(t, J=7.3 Hz, 6H). LCMS (m/z [M+H]⁺): 365.2, Rt₁=0.59 min.

Example 331:N3-(2-(2-fluoropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine

The title compound was synthesized in analogy of Example 111 in 2 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1) and fromN³-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand (2-fluoropyridin-4-yl)boronic acid (CAS no. 401815-98-3, step 2).

1H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 9.18 (s, 1H), 8.69 (d, J=5.5Hz, 1H), 8.46 (d, J=5.2 Hz, 1H), 8.26 (dd, J=4.7, 2.0 Hz, 1H), 7.92 (s,1H), 7.85 (d, J=5.6 Hz, 1H), 2.26 (s, 6H), 2.00 (t, J=6.4 Hz, 2H), 1.66(s, 6H), remark: one CH2 signal is overlapping with DMSO signal. LCMS(m/z [M+H]⁺): 355.2, Rt₁=0.67 min.

Example 332:N³-(2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine

The title compound was synthesized in analogy of Example 111 in 2 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1) and fromN³-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand tert-butyl3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate(CAS no. 1073354-70-7, step 2).

1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 9.11 (s, 1H), 8.95 (s, 1H),8.49 (d, J=5.5 Hz, 1H), 7.68 (d, J=5.6 Hz, 1H), 2.56 (s, 6H), 2.27 (s,6H), 1.92 (t, J=6.1 Hz, 2H), 1.61 (s, 6H). remark: one CH2 signal isoverlapping with DMSO signal. LCMS (m/z [M+H]⁺): 354.2, Rt₁=0.50 min.

Example 333:N¹,N¹,3-trimethyl-N³-(2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized in analogy of Example 111 in 2 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1) and fromN3-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-1H-pyrazole(CAS no. 1218790-40-9, step 2).

1H NMR (400 MHz, DMSO-d6) δ 13.80 (s, 1H), 9.25 (s, 1H), 8.96 (s, 1H),8.57 (d, J=5.5 Hz, 1H), 8.46 (s, 1H), 7.75 (d, J=5.6 Hz, 1H), 2.25 (s,6H), 1.93 (t, J=6.3 Hz, 2H), 1.60 (s, 6H). remark: one CH2 signal isoverlapping with DMSO signal. LCMS (m/z [M+H]⁺): 394.3, Rt₁=0.62 min.

Example 334:N³-(2-(2-aminopyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine

The title compound was synthesized in analogy of Example 111 in 3 stepsfrom 2,4-dichloropyrido[3,4-d]pyrimidine (Intermediate 2c) andN¹,N¹,3-trimethylbutane-1,3-diamine (step 1), fromN³-(2-chloropyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamineand tert-butyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carbamateCAS no. 1095708-32-9 (step 2) and Boc-deprotection using TFA (step 3).

1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.11 (s, 1H), 8.62 (d, J=5.6Hz, 1H), 8.07 (d, J=5.3 Hz, 1H), 7.82 (d, J=5.6 Hz, 1H), 7.49 (s, 1H),7.43 (dd, J=5.3, 1.4 Hz, 1H), 6.11 (s, 2H), 2.47 (d, J=8.1 Hz, 2H), 2.24(s, 6H), 1.99 (t, J=6.4 Hz, 2H), 1.66 (s, 6H). LCMS (m/z [M+H]⁺): 352.2,Rt₁=0.48 min.

Example 335:3-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine

The title compound was synthesized in analogy of Example 1 in 2 stepsfrom 4-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidine (Intermediate 1c)and tert-butyl (4-amino-2-methylbutan-2-yl)carbamate (step 1) andBoc-deprotection using TFA (step 2).

1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.80-8.71 (m, 2H), 8.63 (d,J=5.5 Hz, 1H), 8.37-8.31 (m, 2H), 8.07 (d, J=5.5 Hz, 1H), 3.83-3.73 (m,2H), 1.80-1.68 (m, 2H), 1.15 (s, 6H), 3 exchangeable protons. LCMS (m/z[M+H]⁺): 309.2, Rt₁=0.45 min.

Ex-Vivo Cell Population Expansion and Use in Therapy Starting Materialto Prepare an Expanded Population of Cells:

Autologous Method

The seeding population of cells for use in the method of cell populationexpansion to obtain an expanded population of cells may be obtained froma recipient himself/herself. In patients where tissue, organ, or celldeficiency is partial, for example healthy cells are present, theseeding population of cells may be obtained from non-affected tissue ororgan or cell source. For example, in the case of unilateral ocular celldeficiency, the seeding population may be obtained from a biopsy on thenon-affected eye. It may also be obtained from healthy tissue remainingin an organ that is partially damaged.

Allogenic Method

In a preferred embodiment, the seeding population of cells for use inthe method of cell population expansion to obtain an expanded populationof cells may be obtained from cells originally derived from donor tissue(e.g. human, rabbit, monkey etc, preferably human). For example a sourceof human tissue is from cadaveric donors or tissues from living donors,including living relatives.

From autologous or allogenic tissue derived as described above underautologous and allogenic methods which has been removed from the body,the cells may, for example, be extracted and prepared as follows: Thedesired area may be dissected, for example, using scalpels and the cellsthen dissociated (e.g. using collagenase, dispase, trypsin, accutase orTripLE; for example 1 mg/ml collagenase at 37° C.), until celldetachment becomes apparent by microscopic observation (e.g. using aZeiss Axiovert inverted microscope) from 45 minutes to 3 hours.

For use in the cell population expansion method according to theinvention the isolated cells are then added to medium, for example bypipetting, as described below in the section “Cell populationexpansion”.

In a preferred embodiment according to the invention, an assessment ofthe quality of cellular material harvested from the donor is performed.For example, approximately 24 hours after harvesting the cells andbeginning culturing in medium (growth or cell proliferation medium, asdescribed below), a visual assessment under brightfield microscope tolook for floating cells present (as an indicator of dead cells) may beperformed. Ideally this assessment is to show that there isapproximately less than 10% as floating cells for the material to besuitable for use to generate an expanded population of cells accordingto the invention.

The number of cells suitable for use in the method of cell populationexpansion according to the invention is not limited, but as an examplefor illustrative purposes, the seeding cell population suitable for usein the method of cell population expansion according to the inventionmay comprise approximately 1000 cells.

If it is desired to measure the cell numbers in the seeding cellpopulation, this may be done for example by manual or automated cellcounting using a light microscope, immunohistochemistry or FACSaccording to standard protocols well known in the art.

Ex-Vivo Ocular Cell Population Expansion and Use in Therapy

Described below in more detail is a description of the methodologyrelating to expansion of ocular cell populations (preparation ofstarting material, followed by cell population expansion phase, storageof cells) as applied to ocular cells with the specific examples oflimbal stem cells and corneal endothelial cells.

Starting Material to Prepare an Expanded Population of Limbal StemCells: Corneal Epithelial and Limbal Cells

Autologous Method

The seeding population of cells for use in the method of cell populationexpansion to obtain an expanded population of limbal stem cells may beobtained from the recipient himself/herself. In patients where limbalstem cell deficiency is partial, the seeding population of cells may beobtained from non-affected parts of the limbus. For example, in the caseof unilateral limbal stem cell deficiency, the seeding population may beobtained from a biopsy on the non-affected eye. It may also be obtainedfrom healthy tissue remaining in a limbus that is partially damaged.

Allogenic Method

In a preferred embodiment, the seeding population of cells for use inthe method of cell population expansion to obtain an expanded populationof limbal stem cells may be obtained from cells originally derived fromdonor mammalian corneal tissue (e.g. human, rabbit, monkey etc,preferably human).

For example a source of human corneal tissue is from cadaveric donors(for example sourced through eye banks) or tissues from living donors,including living relatives. A range of donor limbal tissue is suitablefor use according to the invention. In a preferred embodiment limbaltissue is obtained from living relatives or donors with a compatible HLAprofile.

The tissue that is used to obtain the LSCs may, for example, be a ringof limbal tissue of approximately 4 mm in width and 1 mm in height.

From the corneal tissue as described above under autologous andallogenic methods which has been removed from the body, the LSCs may,for example, be extracted and prepared as follows: The limbal epithelialarea may be dissected, for example, using scalpels and the cells thendissociated (e.g. using collagenase, dispase, trypsin, accutase orTripLE; for example 1 mg/ml collagenase at 37° C.), until celldetachment becomes apparent by microscopic observation (e.g. using aZeiss Axiovert inverted microscope) from 45 minutes to 3 hours.

For use in the cell population expansion method according to theinvention the isolated cells are then added to medium, for example bypipetting, as described below in the section “Cell populationexpansion”.

In a preferred embodiment according to the invention, an assessment ofthe quality of cellular material harvested from the donor cornea isperformed. For example, approximately 24 hours after harvesting thecells and beginning culturing in medium (growth or cell proliferationmedium, as described below), a visual assessment under brightfieldmicroscope to look for floating cells present (as an indicator of deadcells) may be performed. Ideally this assessment is to show that thereis approximately less than 10% as floating cells for the material to besuitable for use to generate an expanded population of cells accordingto the invention.

The number of cells suitable for use in the method of cell populationexpansion according to the invention is not limited, but as an examplefor illustrative purposes, the seeding cell population suitable for usein the method of cell population expansion according to the inventionmay comprise approximately 1000 limbal stem cells.

If it is desired to measure the cell numbers in the seeding cellpopulation, this may be done for example by manual or automated cellcounting using a light microscope, immunohistochemistry or FACSaccording to standard protocols well known in the art.

Starting Material to Prepare an Expanded Population of CornealEndothelial Cells

The seeding population of corneal endothelial cells (CECs) for use inthe method of cell population expansion may be obtained from cellsoriginally derived from mammalian corneal tissue (e.g. human, rabbit,monkey etc, preferably human). For example, a source of human cornealtissue is from cadaveric human donors (which may be sourced through eyebanks).

The age of the donors can range, for example, from infancy to 70 yearsof age. Preferably also suitable donors are those who have no history ofcorneal disease or trauma. In one embodiment according to the invention,preferred donor corneas are those where the corneal endothelial cellcount is above 2000 cells/mm² (area). In a more preferred embodimentaccording to the invention the corneal endothelial cell count is 2000 to3500 cells/mm² (area). This is measured for example by examining thecornea of the donor material under a direct light microscope or aspecular microscope as per standard Eye Bank techniques known in the artfor evaluation of donor tissue before transplantation to patients (seeTran et al (2016) Comparison of Endothelial Cell Measurements by Two EyeBank Specular Micorscopes; International Journal of Eye Banking; vol 4.,no 2; 1-8, which is herein incorporated by reference).

The surface of cornea that is used to obtain the CECs is not limited,but may, for example, be an area of approx. 8-10 mm in diameter.

The CECs may, for example, be extracted and prepared as follows from thedonor corneal tissue: The corneal endothelial cell layer and Descemet'smembrane (DM) are scored, for example with a surgical-grade reverseSinsky endothelial stripper. The DM-endothelial cell layer is peeled offthe corneal stroma and cells are dissociated from the DM, for exampleusing 1 mg/ml collagenase at 37° C. until cell detachment becomesapparent by microscopic observation (e.g. using a Zeiss Axiovertinverted microscope) (from 45 minutes to 3 hours). As the DM onlycarries corneal endothelial cells in the cornea, the cell populationisolated in this manner is a population of CECs, which is suitable foruse as a seeding population of cells according to the invention.

For use in the method of cell population expansion according to theinvention the isolated corneal endothelial cells may be added to mediumas described below in the section “Cell population expansion”.

In a preferred embodiment according to the invention, an assessment ofthe quality of cellular material harvested from the donor cornea isperformed. For example, approximately 24 hours after harvesting thecells and beginning culturing in medium (growth or cell proliferationmedium, as described below), a visual assessment under brightfieldmicroscope to look for floating cells present (as an indicator of deadcells) may be performed. Ideally this assessment is to show that thereis approximately less than 10% as floating cells for the material to besuitable for use to generate an expanded population of cells accordingto the invention.

The starting number of cells suitable for use in the method of cellpopulation expansion according to the invention is not limited, but asan example for illustrative purposes, the seeding population of cornealendothelial cells suitable for use in the method of cell populationexpansion according to the invention may be 100000 to 275000 cells.

If it is desired to measure the cell numbers in the seeding cellpopulation, this may be done for example by taking an aliquot andperforming immunocytochemistry (e.g. to count nuclei stained with SytoxOrange) or by live cell imaging under brightfield microscope to countthe number of cells.

The Sytox Orange assay may be performed according to standard protocolsknown in the art. In brief, after cells have attached to the cellculture dish (typically 24 h after cell plating), the cells are fixed inparaformaldehyde. The cells are then permeabilized (e.g. using asolution of 0.3% Triton X-100) and they are then labeled in a solutionof Sytox Orange (e.g. using 0.5 micromolar of Sytox Orange in PBS). Thenumber of nuclei stained with Sytox Orange per surface area are thencounted under a Zeiss epifluorescence microscope.

Cell Population Expansion

In one embodiment of the invention, a population of cells comprisingcells from a patient or a donor, can be grown in medium in a culturecontainer known in the art, such as plates, multi-well plates, and cellculture flasks. For example, a culture dish may be used which isnon-coated or coated with collagen, synthemax, gelatin or fibronectin. Apreferred example of a suitable culture container is a non-coated plate.Standard culturing containers and equipment such as bioreactors known inthe art for industrial use may also be used.

The medium used may be a growth medium or a cell proliferation medium.In general, a growth medium is a culture medium supporting the growthand maintenance of a population of cells. Those of skill in art canreadily determine an appropriate growth medium for a particular type ofcell population. Suitable growth mediums are known in the art for stemcell culture or epithelial cell culture are for example: DMEM(Dulbecco's Modified Eagle's Medium) supplemented with FBS (Fetal BovineSerum) (Invitrogen), human endothelial SF (serum free) medium(Invitrogen) supplemented with human serum, X-VIVO15 medium (Lonza), orDMEM/F12 (Thermo Fischer Scientific) (optionally supplemented withcalcium chloride). These may be additionally supplemented with growthfactors (e.g. bFGF), and/or antibiotics such as penicillin andstreptomycin.

Alternatively, isolated cells may be added first to a cell proliferationmedium according to the invention. The cell proliferation medium asdefined herein comprises a growth medium and a LATS inhibitor accordingto the invention.

In certain embodiments, a cell proliferation medium of the inventioncomprises a growth medium and a LATS inhibitor according to theinvention. The LATS inhibitor is preferably selected from the groupcomprising compounds according to Formula A1 or subformulae thereof andas further described under the section “LATS inhibitors”.

In a preferred embodiment the LATS inhibitors according to Formula A1 orsubformulae thereof are added at a concentration of about 0.5 to 100micromolar, preferably about 0.5 to 25 micromolar, more preferably about1 to 20 micromolar. In a specific embodiment the LATS inhibitorsaccording to Formula A1 or subformulae thereof are added at aconcentration of about 3 to 10 micromolar.

In one embodiment, the stock solution of the compound according toFormula A1 or subformulae thereof may be prepared by dissolving thecompound powder to a stock concentration of 10 mM in DMSO.

In one aspect of the invention the LATS inhibitor according to theinvention inhibits LATS1 and/or LATS2 activity in the cell population.In a preferred embodiment the LATS inhibitor inhibits LATS1 and LATS2.

The cells may go through a round or rounds of addition of fresh growthmedium and/or cell proliferation medium. The cells do not need to bepassaged in order for fresh medium to be added, but passaging cells isalso a way to add fresh medium.

A series of mediums may be also used, in various combinations of orders:for example a cell proliferation medium, followed by addition of agrowth medium (which is not supplemented with LATS inhibitors accordingto the invention, and may be different to the growth medium used as thebase for the cell proliferation medium).

The cell population expansion phase according to the invention occursduring the period the cells are exposed to the cell proliferationmedium.

Standard temperature conditions known in the art for culturing cells maybe used, for example preferably about 30° C. to 40° C. Particularlypreferably cell growth, as well as the cell population expansion phaseis carried out at about 37° C. A conventional cell incubator with 5-10%CO₂ levels may be used. Preferably the cells are exposed to 5% CO₂.

The cells may be passaged during the culturing in the growth or cellproliferation medium as necessary. Cells may be passaged when they aresub-confluent or confluent. Preferably the cells are passaged when theyreach approximately 90%-100% confluency, although lower percentageconfluency levels may also be performed. The passaging of cells is doneaccording to standard protocols known in the art. For example, in briefcells are passaged by treating cultures with Accutase (e.g. for 10minutes), rinsing the cell suspension by centrifugation and platingcells in fresh growth medium or cell proliferation medium as desired.Cell splitting ratios range, for example, from 1:2 to 1:5.

For the cell population expansion phase of the method of cell populationexpansion according to the invention, the expansion of the seeding cellpopulation in the cell proliferation medium may be performed until therequired amount of cellular material is obtained.

The cells may be exposed to the cell proliferation medium for a range oftime periods in order to expand the cell population.

In a preferred embodiment the seeding cell population is exposed to theLATS inhibitors according to the invention (such as those compoundsaccording to Formula A1 or subformulae thereof) directly after cellisolation from the patient or donor tissue and maintained for the entiretime that cell proliferation is required, for example 12 to 16 days.

In one embodiment according to the invention, a gene editing techniquemay optionally be performed to genetically modify cells and/or toexpress a biotherapeutic compound. For example, the cells may bemodified to reduce or eliminate the expression and/or function of animmune response mediating gene, which may otherwise contribute to immunerejection when the cell population is delivered to the patient. Theapplication of gene editing techniques in the method of cell populationexpansion according to the invention is optional, and the administrationto the patient of topical immunosuppressants and/or anti-inflammatoryagents (as described further under the section Immunosuppressant andAnti-inflammatory agent) may instead be used if desired to mitigateissues with immunorejection of the transplanted material in the patient.

According to one aspect of the invention, genetically modifyingcomprises reducing or eliminating the expression and/or function of agene associated with facilitating a host versus graft immune response.In a preferred embodiment, genetically modifying comprises introducinginto an isolated stem cell or stem cell population a gene editing systemwhich specifically targets a gene associated with facilitating a hostversus graft immune response. In a specific embodiment, said geneediting system is selected from the group consisting of CRISPR (CRISPR:clustered regularly interspaced short palindromic repeats, also known asCRISPR/Cas systems), ZFN (Zinc-finger nucleases), TALEN (transcriptionactivator-like effector based nucleases), engineered meganucleases (e.g.ARCUS nucleases, such as the ARC nuclease), AAV vector (adeno-associatedvirus) and lentiviral vectors-based genome editing technologies.

A gene editing technique, if it is to be used, may be performed atdifferent points, such as for example (1) on tissue, before cellisolation or (2) at the time of cell isolation or (3) during the cellpopulation expansion phase in vitro (when the cells are exposed to aLATS inhibitor according to the invention in vitro) or (4) in vitro atthe end of the cell population expansion phase (after the cells areexposed to a LATS inhibitor according to the invention in vitro). In aspecific embodiment, CRISPR is used after two weeks of in vitroexpansion of the cell population in the presence of the LATS inhibitoraccording to the invention.

The gene editing techniques suitable for use in the method of cellpopulation expansion are further described under the section “reductionof immunorejection”.

In the method of cell population expansion according to the inventionthe LATS inhibitors, which are preferably compounds, produce greaterthan 2 fold expansion of the seeded population of cells.

In one aspect of the method of cell population expansion according tothe invention the compounds according to Formula A1 or subformulaethereof produce greater than 30 fold expansion of the seeded populationof isolated cells (i.e., cells obtained from a patient or a donor). In aspecific embodiment of the method of cell population expansion accordingto the invention, the LATS inhibitors according to Formula A1 orsubformulae thereof produce 100 fold to 2200 fold expansion of theseeded population of isolated cells. In a more specific embodiment ofthe method of cell population expansion according to the invention, theLATS inhibitors according to Formula A1 or subformulae thereof produce600 fold to 2200 fold expansion of the seeded population of isolatedcells. The fold expansion factor achieved by the method of cellpopulation expansion according to the invention may be achieved in oneor more passages of the cells. In another aspect of the invention thefold expansion factor achieved by the method of cell populationexpansion according to the invention may be achieved after exposure tothe compound according to Formula A1 or subformulae thereof for about 12to 16 days, preferably about 14 days.

If it is desired to measure the cell number or expansion of the cellpopulation, this may be done for example by taking an aliquot andperforming immunocytochemistry (e.g. to count nuclei stained with SytoxOrange) or by live cell imaging under brightfield microscope to countthe number of cells or by performing real-time quantitative live-cellanalysis of cell confluence at various time points during the cellpopulation expansion phase of the method according to the invention.

The Sytox Orange assay may be performed according to standard protocolsknown in the art. In brief, after cells have attached to the cellculture dish (typically 24 h after cell plating), the cells are fixed inparaformaldehyde. The cells are then permeabilized (e.g. using asolution of 0.3% Triton X-100) and they are then labeled in a solutionof Sytox Orange (e.g. using 0.5 micromolar of Sytox Orange in PBS). Thenumber of nuclei stained with Sytox Orange per surface area are thencounted under a Zeiss epifluorescence microscope. The cell populationexpanded by the method of cell population expansion according to theinvention may be added to a solution and then stored, for example in apreservation or cryopreservation solution (such as those describedbelow), or added directly to a composition suitable for delivery to apatient. The preservation, cryopreservation solution or compositionsuitable for ocular delivery may optionally comprise a LATS inhibitoraccording to the invention.

In a more preferred embodiment according to the invention, the cellpopulation preparation which is delivered to a patient comprises verylow to negligible levels of a LATS inhibitor compound. Thus in aspecific embodiment, the method of cell population expansion accordingto the invention comprises the further step of rinsing to substantiallyremove the compound according to the invention (such as the compoundaccording to Formula A1 or subformulae thereof). This may involverinsing the cells after the cell population expansion phase according tothe invention. To rinse the cells, the cells are detached from theculture dish (e.g. by treating with Accutase), the detached cells arethen centrifuged, and a cell suspension is made in PBS or growth mediumaccording to the invention. This step may be performed multiple times,e.g. one to ten times, to rinse out the cells. Finally the cells may beresuspended in a preservation solution, cryopreservation solution, acomposition suitable for ocular delivery, growth medium or combinationsthereof as desired.

The expanded population of cells prepared by the method of cellpopulation expansion and rinsed of cell proliferation medium comprisinga LATS inhibitor according the invention may be transferred to acomposition suitable for delivery to a patient, such as for example alocalising agent. Optionally the cell population is stored for a periodbefore addition to a localising agent suitable for delivery to apatient. In a preferred embodiment, the expanded cell population mayfirst be added to a solution suitable for preservation orcryopreservation, which preferably does not comprise a LATS inhibitor,and the cell population stored (optionally with freezing) beforeaddition to a localising agent suitable for delivery to a patient, whichalso preferably does not comprise a LATS inhibitor.

Typical solutions suitable for cryopreservation, glycerol, dimethylsulfoxide, propylene glycol or acetamide may be used in thecryopreservation solution of the present invention. The cryopreservedpreparation of cells is typically kept at −20° C. or −80° C.

Cell Population Expansion: To Prepare an Expanded Population of LimbalStem Cells

In one embodiment of the invention, a population of cells comprisingcorneal epithelial and limbal cells, including limbal stem cells, forexample obtained as described in the section “Starting material toprepare an expanded population of limbal stem cells: Corneal epithelialand limbal cells”, can be grown in medium in a culture container knownin the art, such as plates, multi-well plates, and cell culture flasks.For example, a culture dish may be used which is non-coated or coatedwith collagen, synthemax, gelatin or fibronectin. A preferred example ofa suitable culture container is a non-coated plate. Standard culturingcontainers and equipment such as bioreactors known in the art forindustrial use may also be used.

The medium used may be a growth medium or a cell proliferation medium. Agrowth medium is defined herein as a culture medium supporting thegrowth and maintenance of a population of cells. Suitable growth mediumsare known in the art for stem cell culture or epithelial cell cultureare for example: DMEM (Dulbecco's Modified Eagle's Medium) supplementedwith FBS (Fetal Bovine Serum) (Invitrogen), human endothelial SF (serumfree) medium (Invitrogen) supplemented with human serum, X-VIVO15 medium(Lonza), or DMEM/F12 (Thermo Fischer Scientific) (optionallysupplemented with calcium chloride). These may be additionallysupplemented with growth factors (e.g. bFGF), and/or antibiotics such aspenicillin and streptomycin. A preferred growth medium according to theinvention is X-VIVO15 medium (which is not additionally supplementedwith growth factors).

Alternatively, the isolated cells may be added first to a cellproliferation medium according to the invention. The cell proliferationmedium as defined herein comprises a growth medium and a LATS inhibitoraccording to the invention. In the cell proliferation medium accordingto the invention the growth medium component is selected from the groupconsisting of DMEM (Dulbecco's Modified Eagle's Medium) supplementedwith FBS (Fetal Bovine Serum) (Invitrogen), human endothelial SF (serumfree) medium (Invitrogen) supplemented with human serum, X-VIVO15 medium(Lonza or DMEM/F12 (Thermo Fischer Scientific) (optionally supplementedwith calcium chloride). These may be additionally supplemented withgrowth factors (e.g. bFGF), and/or antibiotics such as penicillin andstreptomycin.

A preferred cell proliferation medium according to the invention isX-VIVO15 medium (Lonza) with a LATS inhibitor according to theinvention. This cell proliferation medium has the advantage that it doesnot need additional growth factors or feeder cells to facilitate theproliferation of the LSCs. X-VIVO medium comprises inter aliapharmaceutical grade human albumin, recombinant human insulin, andpasteurized human transferrin. Optionally antibiotics may be added toX-VIVO15 medium. In a preferred embodiment, X-VIVO15 medium is usedwithout the addition of antibiotics.

The cell proliferation medium comprises a growth medium and a LATSinhibitor according to the invention. The LATS inhibitor is preferablyselected from the group comprising compounds according to Formula A1 orsubformulae thereof and as further described under the section “LATSinhibitors”.

In a preferred embodiment the LATS inhibitors according to Formula A1 orsubformulae thereof are added at a concentration of about 0.5 to 100micromolar, preferably about 0.5 to 25 micromolar, more preferably about1 to 20 micromolar. In a specific embodiment the LATS inhibitorsaccording to Formula A1 or subformulae thereof are added at aconcentration of about 3 to 10 micromolar.

In one embodiment, the stock solution of the compound according toFormula A1 or subformulae thereof may be prepared by dissolving thecompound powder to a stock concentration of 10 mM in DMSO.

In one aspect of the invention the LATS inhibitor according to theinvention inhibits LATS1 and/or LATS2 activity in the limbal cells. In apreferred embodiment the LATS inhibitor inhibits LATS1 and LATS2.

The cells may go through a round or rounds of addition of fresh growthmedium and/or cell proliferation medium. The cells do not need to bepassaged in order for fresh medium to be added, but passaging cells isalso a way to add fresh medium.

A series of mediums may be also used, in various combinations of orders:for example a cell proliferation medium, followed by addition of agrowth medium (which is not supplemented with LATS inhibitors accordingto the invention, and may be different to the growth medium used as thebase for the cell proliferation medium).

The cell population expansion phase according to the invention occursduring the period the cells are exposed to the cell proliferationmedium.

Standard temperature conditions known in the art for culturing cells maybe used, for example preferably about 30° C. to 40° C. Particularlypreferably cell growth, as well as the cell population expansion phaseis carried out at about 37° C. A conventional cell incubator with 5-10%CO₂ levels may be used. Preferably the cells are exposed to 5% CO₂.

The cells may be passaged during the culturing in the growth or cellproliferation medium as necessary. Cells may be passaged when they aresub-confluent or confluent. Preferably the cells are passaged when theyreach approximately 90%-100% confluency, although lower percentageconfluency levels may also be performed. The passaging of cells is doneaccording to standard protocols known in the art. For example, in briefcells are passaged by treating cultures with Accutase (e.g. for 10minutes), rinsing the cell suspension by centrifugation and platingcells in fresh growth medium or cell proliferation medium as desired.Cell splitting ratios range, for example, from 1:2 to 1:5.

For the cell population expansion phase of the method of cell populationexpansion according to the invention, the expansion of the seeding cellpopulation in the cell proliferation medium may be performed until therequired amount of cellular material is obtained.

The cells may be exposed to the cell proliferation medium for a range oftime periods in order to expand the cell population. For example thismay include the entire time that the LSCs are kept in culture, or forthe first week after LSC isolation or for 24 hours after dissection ofthe limbus from the cornea.

In a preferred embodiment the seeding cell population is exposed to theLATS inhibitors according to the invention (such as those compoundsaccording to Formula A1 or subformulae thereof) directly after cellisolation from the cornea and maintained for the entire time that LSCproliferation is required, for example 12 to 16 days.

In one embodiment according to the invention, a gene editing techniquemay optionally be performed to genetically modify cells, to reduce oreliminate the expression and/or function of an immune response mediatinggene which may otherwise contribute to immune rejection when the cellpopulation is delivered to the patient. The application of gene editingtechniques in the method of cell population expansion according to theinvention is optional, and the administration to the patient of topicalimmunosuppressants and/or anti-inflammatory agents (as described furtherunder the section Immunosuppressant and Anti-inflammatory agent) mayinstead be used if desired to mitigate issues with immunorejection ofthe transplanted material in the patient.

According to one aspect of the invention, genetically modifyingcomprises reducing or eliminating the expression and/or function of agene associated with facilitating a host versus graft immune response.In a preferred embodiment, genetically modifying comprises introducinginto a limbal stem cell a gene editing system which specifically targetsa gene associated with facilitating a host versus graft immune response.In a specific embodiment, said gene editing system is selected from thegroup consisting of CRISPR (CRISPR: clustered regularly interspacedshort palindromic repeats, also known as CRISPR/Cas systems), ZFN(Zinc-finger nucleases), TALEN (transcription activator-like effectorbased nucleases), engineered meganucleases (e.g. ARCUS nucleases, suchas the ARC nuclease), AAV vector (adeno-associated virus) gene editing(e.g., AAV vector driven homologous recombination) and lentiviralvectors-based genome editing technologies. AAV vector driven genedelivery, such as driven by homologous recombination, can be achieved byan AAV selected from the group consisting of: AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, or a derivative thereof.

A gene editing technique, if it is to be used, may be performed atdifferent points, such as for example (1) on limbal epithelial tissue,before LSC isolation or (2) at the time of cell isolation or (3) duringthe cell population expansion phase in vitro (when the cells are exposedto a LATS inhibitor according to the invention in vitro) or (4) in vitroat the end of the cell population expansion phase (after the cells areexposed to a LATS inhibitor according to the invention in vitro). In aspecific embodiment CRISPR is used after two weeks of in vitro expansionof the cell population in the presence of the LATS inhibitor accordingto the invention.

The gene editing techniques suitable for use in the method of cellpopulation expansion are further described under the section “reductionof immunorejection”.

In the method of cell population expansion according to the inventionthe LATS inhibitors, which are preferably compounds, produce greaterthan 2 fold expansion of the seeded population of cells.

In one aspect of the method of cell population expansion according tothe invention the compounds according to Formula A1 or subformulaethereof produce greater than 30 fold expansion of the seeded populationof limbal cells. In a specific embodiment of the method of cellpopulation expansion according to the invention, the LATS inhibitorsaccording to Formula A1 or subformulae thereof produce 100 fold to 2200fold expansion of the seeded population of limbal cells. In a morespecific embodiment of the method of cell population expansion accordingto the invention, the LATS inhibitors according to Formula A1 orsubformulae thereof produce 600 fold to 2200 fold expansion of theseeded population of limbal cells. The fold expansion factor achieved bythe method of cell population expansion according to the invention maybe achieved in one or more passages of the cells. In another aspect ofthe invention the fold expansion factor achieved by the method of cellpopulation expansion according to the invention may be achieved afterexposure to the compound according to Formula A1 or subformulae thereoffor about 12 to 16 days, preferably about 14 days.

In one aspect of the method of cell population expansion according tothe invention, the LATS inhibitors according to Formula A1 orsubformulae thereof produce a cell population with more than 6% ofp63alpha positive cells compared to the total amount of cells. In aspecific embodiment of the method of cell population expansion accordingto the invention, the LATS inhibitors according to Formula A1 orsubformulae thereof produce a cell population with more than 20% ofp63alpha positive cells compared to the total amount of cells. Inanother specific embodiment of the method of cell population expansionaccording to the invention, the LATS inhibitors according to Formula A1or subformulae thereof produce a cell population with more than 70% ofp63alpha positive cells compared to the total amount of cells. In yetanother specific embodiment of the method of cell population expansionaccording to the invention the LATS inhibitors according to Formula A1or subformulae thereof produce a cell population with more than 95% ofp63alpha positive cells compared to the total amount of cells. Theincrease in the percentage of p63alpha positive cells achieved by themethod of cell population expansion according to the invention may beachieved in one or more passages of the cells. In another aspect of theinvention the increase in the percentage of p63alpha positive cellsachieved by the method of cell population expansion according to theinvention may be achieved after exposure to the compound according toFormula A1 or subformulae thereof for about 12 to 16 days, preferablyabout 14 days.

If it is desired to measure the cell number or expansion of the cellpopulation, this may be done for example by taking an aliquot andperforming immunocytochemistry (e.g. to count nuclei stained with SytoxOrange) or by live cell imaging under brightfield microscope to countthe number of cells or by performing real-time quantitative live-cellanalysis of cell confluence at various time points during the cellpopulation expansion phase of the method according to the invention.

The Sytox Orange assay may be performed according to standard protocolsknown in the art. In brief, after cells have attached to the cellculture dish (typically 24 h after cell plating), the cells are fixed inparaformaldehyde. The cells are then permeabilized (e.g. using asolution of 0.3% Triton X-100) and they are then labeled in a solutionof Sytox Orange (e.g. using 0.5 micromolar of Sytox Orange in PBS). Thenumber of nuclei stained with Sytox Orange per surface area are thencounted under a Zeiss epifluorescence microscope.

In one aspect according to the invention the LSC population obtainableor obtained by the method of cell population expansion according to theinvention preferably shows at least one of the followingcharacteristics. More preferably, it shows two or more, more preferablyall, of the following characteristics.

-   (1) The cell preparation is positive for p63alpha cells. The    expression of p63alpha may be estimated by standard techniques known    in the art, such as for example immunohistochemistry and    quantitative RT-PCR.-   (2) The cell preparation comprises more than 6% p63alpha positive    cells. Preferably the cell preparation comprises more than 10%, 20%,    30%, 40%, 50%, 60%, 70%, 80% or 90% p63alpha positive cells. In a    preferred embodiment the cell preparation comprises more than 95%    p63alpha positive cells. The percentage of p63alpha cells may be    measured by immunohistochemistry or FACS.-   (3) The cells express one or more of ABCB5, ABCG2, and C/EBPb. The    expression of ABCB5, ABCG2, and C/EBPb may be estimated by standard    techniques known in the art, such as for example    immunohistochemistry and quantitative RT-PCR.-   (4) The cells can differentiate into corneal epithelium cells as    observed by keratin-12 expression. These characteristics can be    observed by immunohistochemistry or FACS.

The cell population expanded by the method of cell population expansionaccording to the invention may be added to a solution and then stored,for example in a preservation or cryopreservation solution (such asthose described below), or added directly to a composition suitable forocular delivery. The preservation, cryopreservation solution orcomposition suitable for ocular delivery may optionally comprise a LATSinhibitor according to the invention.

In a more preferred embodiment according to the invention, the cellpopulation preparation which is delivered to the eye comprises very lowto neglible levels of a LATS inhibitor compound. Thus in a specificembodiment, the method of cell population expansion according to theinvention comprises the further step of rinsing to substantially removethe compound according to the invention (such as the compound accordingto Formula A1 or subformulae thereof). This may involve rinsing thecells after the cell population expansion phase according to theinvention. To rinse the cells, the cells are detached from the culturedish (e.g. by treating with Accutase), the detached cells are thencentrifuged, and a cell suspension is made in PBS or growth mediumaccording to the invention. This step may be performed multiple times,e.g. one to ten times, to rinse out the cells. Finally the cells may beresuspended in a preservation solution, cryopreservation solution, acomposition suitable for ocular delivery, growth medium or combinationsthereof as desired.

The expanded population of cells prepared by the method of cellpopulation expansion and rinsed of cell proliferation medium comprisinga LATS inhibitor according the invention may be transferred to acomposition suitable for ocular delivery, such as for example alocalising agent. Optionally the cell population is stored for a periodbefore addition to a localising agent suitable for ocular delivery. In apreferred embodiment, the expanded cell population may first be added toa solution suitable for preservation or cryopreservation, whichpreferably does not comprise a LATS inhibitor, and the cell populationstored (optionally with freezing) before addition to a localising agentsuitable for ocular delivery, which also preferably does not comprise aLATS inhibitor.

Typical solutions suitable for preservation of LSCs are Optisol or PBS,preferably Optisol. Optisol is a corneal storage medium comprisingchondroitin sulfate and dextran to enhance corneal dehydration duringstorage (see for example Kaufman et al., (1991) Optisol corneal storagemedium; Arch Ophthalmol June; 109(6): 864-8). For cryopreservation,glycerol, dimethyl sulfoxide, propylene glycol or acetamide may be usedin the cryopreservation solution of the present invention. Thecryopreserved preparation of cells is typically kept at −20° C. or −80°C.

In one aspect the invention relates to a preserved or cryopreservedpreparation of limbal stem cells obtainable by the method of cellpopulation expansion according to the invention. In an alternativeaspect the invention relates to a fresh cell preparation where limbalstem cells obtainable by the method of cell population expansionaccording to the invention are in suspension in PBS and/or growth mediumor combined with a localising agent. The fresh cell preparation istypically kept at about 15 to 37° C. Standard cell cultures containersknown in the art may be used to store the cells, such as a vial or aflask.

In a preferred embodiment according to the invention, before use in theeye, a cryopreserved preparation of cells is thawed (for example byincubating at a temperature of about 37° C. in an incubator orwaterbath). Preferably 10 volumes of PBS or growth medium may be addedto rinse off the cells from the cryopreservant solution. Cells may thenbe rinsed by centrifugation, and a cell suspension may be made in PBSand/or growth medium, before combination with a localising agent forocular delivery, which also preferably does not comprise a LATSinhibitor.

In one aspect of the invention the expanded population of cells preparedby the method of cell population expansion, are prepared as a suspension(for example in PBS and/or growth medium, such as for example X-VIVOmedium) and combined with a localising agent suitable for oculardelivery, (such as a biomatrix like GelMA). In a specific embodiment ofthe method of treatment according to the invention, this combination ofcells, PBS and/or growth medium, and biomatrix is delivered to the eyevia a carrier (such as a contact lens).

In yet another specific embodiment this combination of cells, PBS and/orgrowth medium, and biomatrix comprises at most only trace levels of aLATS inhibitor.

The term “trace levels” as used herein means less than 5% w/v (e.g., nomore than 5% w/v, 4% w/v, 3% w/v, 2% w/v, or 1% w/v), and preferablyless than 0.01% w/v (e.g., no more than 0.01% w/v, 0.009% w/v, 0.008%w/v, 0.007% w/v, 0.006% w/v, 0.005% w/v, 0.004% w/v, 0.003% w/v, 0.002%w/v, or 0.001% w/v), which can be measured, for example usinghigh-resolution chromatography as described in the Examples herein. Incertain embodiments, trace levels of a LATS inhibitor compound of theinvention are the levels of residual compounds present after one or morewash steps, which collectively are below the cellular potency of suchcompounds, and accordingly they do not induce biological effect in vivo.Accordingly, residual levels of compounds are below the amount expectedto have a biological effect on cell population expansion in cell cultureor in a subject (e.g., after transplantation of an expanded cellpopulation to the subject). Trace levels can be measured, for example,as the wash-off efficiency, which can be calculated as follows: Wash-offefficiency=100−(average concentration in post-wash pellet x pelletvolume x molecule weight)/(compound concentration×culture media volume xmolecule weight). As used herein, “rinsing to substantially remove” aLATS inhibitor compound of the invention from cells refers to steps forestablishing trace levels of the LATS inhibitor compound.

Alternatively, the cells may be cultured and the cell populationproliferation phase may occur in cell proliferation medium on alocalising agent suitable for cell delivery to the ocular surface (forexample fibrin, collagen).

Cell Population Expansion: To Prepare an Expanded Population of CornealEndothelial Cells

In a preferred embodiment of the invention, corneal endothelial cells,for example isolated and obtainable as described in the section“Starting material to prepare an expanded population of cornealendothelial cells”, can be grown in medium in a culture container knownin the art, such as plates, multi-well plates, and cell culture flasks.For example, a culture dish may be used which is non-coated or coatedwith collagen, synthemax, gelatin or fibronectin. A preferred example ofa suitable culture container is a non-coated plate.

Standard culturing containers and equipment such as bioreactors known inthe art for industrial use may also be used.

The medium used may be a growth medium or a cell proliferation medium. Agrowth medium is defined herein as a culture medium supporting thegrowth and maintenance of a population of cells. Suitable growth mediumsare known in the art for corneal endothelial cell culture are forexample: DMEM (Dulbecco's Modified Eagle's Medium) supplemented with FBS(Fetal Bovine Serum) (Invitrogen), human endothelial SF (serum free)medium (Invitrogen) supplemented with human serum, X-VIVO15 medium(Lonza) or mesenchymal stem cell-conditioned medium. These may beadditionally supplemented with growth factors (e.g. bFGF), and/orantibiotics such as penicillin and streptomycin. A preferred growthmedium according to the invention is X-VIVO15 medium (which is notadditionally supplemented with growth factors).

Alternatively, the isolated cells may be added first to a cellproliferation medium according to the invention. The cell proliferationmedium as defined herein comprises a growth medium and a LATS inhibitoraccording to the invention. In the cell proliferation medium accordingto the invention the growth medium component is selected from the groupconsisting of DMEM (Dulbecco's Modified Eagle's Medium) supplementedwith FBS (Fetal Bovine Serum) (Invitrogen), human endothelial SF (serumfree) medium (Invitrogen) supplemented with human serum, X-VIVO15 medium(Lonza) or mesenchymal stem cell-conditioned medium. These may beadditionally supplemented with growth factors (e.g. bFGF), and/orantibiotics such as penicillin and streptomycin.

A preferred cell proliferation medium according to the invention isX-VIVO15 medium (Lonza) with a LATS inhibitor according to theinvention. This cell proliferation medium has the advantage that it doesnot need additional growth factors or feeder cells to facilitate theproliferation of the CECs. X-VIVO medium comprises inter aliapharmaceutical grade human albumin, recombinant human insulin, andpasteurised human transferrin. Optionally antibiotics may be added toX-VIVO15 medium. In a preferred embodiment, X-VIVO15 medium is usedwithout the addition of antibiotics.

The cell proliferation medium comprises a growth medium and a LATSinhibitor according to the invention. The LATS inhibitor is preferablyselected from the group comprising compounds according to Formula A1 orsubformulae thereof and as further described under the section “LATSInhibitors”.

In a preferred embodiment the LATS inhibitors according to Formula A1 orsubformulae thereof are added at a concentration of about 0.5 to 100micromolar, preferably about 0.5 to 25 micromolar, more preferably about1 to 20 micromolar. In a specific embodiment the LATS inhibitorsaccording to Formula A1 or subformulae thereof are added at aconcentration of about 3 to 10 micromolar.

In one embodiment, the stock solution of the compound according toFormula A1 or subformulae thereof may be prepared by dissolving thecompound powder to a stock concentration of 10 mM in DMSO.

In one aspect of the invention the LATS inhibitor according to theinvention inhibits LATS1 and/or LATS2 activity in the cornealendothelial cells. In a preferred embodiment the LATS inhibitor inhibitsLATS1 and LATS2.

The cells may go through a round or rounds of addition of fresh growthmedium and/or cell proliferation medium. The cells do not need to bepassaged in order for fresh medium to be added, but passaging cells isalso a way to add fresh medium.

A series of mediums may be also used, in various combinations of orders:for example a cell proliferation medium, followed by addition of agrowth medium (which is not supplemented with LATS inhibitors accordingto the invention, and may be different to the growth medium used as thebase for the cell proliferation medium).

The cell population expansion phase according to the invention occursduring the period the cells are exposed to the cell proliferationmedium.

Standard temperature conditions known in the art for culturing cells maybe used, for example preferably about 30° C. to 40° C. Particularlypreferably cell growth, as well as the cell population expansion phaseis carried out at about 37° C. A conventional cell incubator with 5-10%CO₂ levels may be used. Preferably the cells are exposed to 5% CO₂.

The cells may be passaged during the culturing in the growth or cellproliferation medium as necessary. Cells may be passaged when they aresub-confluent or confluent. Preferably the cells are passaged when theyreach approximately 90%-100% confluency, although lower percentageconfluency levels may also be performed. The passaging of cells is doneaccording to standard protocols known in the art. For example, in briefthe cells are detached from the culture container, for example usingcollagenase. The cells are then centrifuged and rinsed in PBS or thecell growth medium according to the invention and plated in fresh growthor cell proliferation medium as desired at a dilution of, for example,1:2 to 1:4.

For the cell population expansion phase of the method of cell populationexpansion according to the invention, the expansion of the seeding cellpopulation in the cell proliferation medium may be performed until therequired amount of cellular material is obtained.

The cells may be exposed to the cell proliferation medium for a range oftime periods in order to expand the cell population. For example, thismay include the entire time that the CECs are kept in culture, or onlyfor the first one to two weeks after CEC isolation or only for 24 hoursafter dissection of the cornea.

In a preferred embodiment, the corneal endothelial cells are exposed tothe LATS inhibitors according to the invention (such as those compoundsaccording to Formula A1 or subformulae thereof) directly after cellisolation from the cornea, and maintained for the entire time that CECproliferation is required, for example one to two weeks.

In a more preferred embodiment of the invention, after the cellpopulation expansion phase in vitro (i.e. after the cells are exposed toa LATS inhibitor according to the invention for a period of time toexpand the population of cells), the method of cell population expansionaccording to the invention comprises a further step wherein the cellsmay be grown for a period of time (e.g. two weeks) in growth mediumwithout supplementation of a LATS inhibitor, to enable a mature cornealendothelium to form. A mature corneal endothelium is defined herein as amonolayer of CECs with hexagonal morphology, ZO-1-positive tightjunctions and expression of Na/K ATPase. In a preferred embodiment thecells are not passaged while the mature corneal endothelium is formed.

In one embodiment according to the invention, a gene editing techniquemay optionally be performed to genetically modify cells, to reduce oreliminate the expression and/or function of an immune response mediatinggene which may otherwise contribute to immune rejection when the cellpopulation is delivered to the patient. The application of gene editingtechniques in the method of cell population expansion according to theinvention is optional, and the administration to the patient of topicalimmunosuppressants and/or anti-inflammatory agents (as described furtherunder the section Immunosuppressant and Anti-inflammatory agent) mayinstead be used if desired to mitigate issues with immunorejection ofthe transplanted material in the patient.

According to one aspect of the invention, for the scenario that a geneediting technique is used, genetically modifying comprises reducing oreliminating the expression and/or function of a gene associated withfacilitating a host versus graft immune response. In a preferredembodiment, genetically modifying comprises introducing into a cornealendothelial cell a gene editing system which specifically targets a geneassociated with facilitating a host versus graft immune response. In aspecific embodiment, said gene editing system is selected from the groupconsisting of CRISPR (CRISPR: clustered regularly interspaced shortpalindromic repeats, also known as CRISPR/Cas systems), ZFN (Zinc-fingernucleases), TALEN (transcription activator-like effector basednucleases), engineered meganucleases (e.g. ARCUS nucleases, such as theARC nuclease), AAV vector (adeno-associated virus) gene editing (e.g.AAV vector driven homologous recombination) and lentiviral vectors-basedgenome editing technologies. AAV vector driven gene delivery, such asdriven by homologous recombination, can be achieved by an AAV selectedfrom the group consisting of: AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, or a derivative thereof.

A gene editing technique, if it is to be used, may be performed atdifferent points, such as for example (1) on corneal tissue, before CECisolation or (2) at the time of cell isolation or (3) during the cellpopulation expansion phase in vitro (when the cells are exposed to aLATS inhibitor according to the invention in vitro) or (4) in vitro atthe end of the cell population expansion phase (after the cells areexposed to a LATS inhibitor according to the invention in vitro).

The gene editing techniques suitable for use in the method of cellpopulation expansion are further described under the section “reductionof immunorejection”.

In the method of cell population expansion according to the inventionthe LATS inhibitors, which are preferably compounds, produce greaterthan 2 fold expansion of the seeded population of cells.

In one aspect of the method of cell population expansion according tothe invention the compounds according to Formula A1 or subformulaethereof produce greater than 10 fold expansion of the seeded populationof corneal endothelial cells. In a specific embodiment of the method ofcell population expansion according to the invention, the LATSinhibitors according to Formula A1 or subformulae thereof produce 15fold to 600 fold expansion of the seeded population of cornealendothelial cells. In a more specific embodiment of the method of cellpopulation expansion according to the invention, the LATS inhibitorsaccording to Formula A1 or subformulae thereof produce 20 fold to 550fold expansion of the seeded population of corneal endothelial cells.The fold expansion factor achieved by the method of cell populationexpansion according to the invention may be achieved in one or morepassages of the cells. In another aspect of the invention the foldexpansion factor achieved by the method of cell population expansionaccording to the invention may be achieved after exposure to thecompound according to Formula A1 or subformulae thereof for one to twoweeks, preferably after about 10 days.

If it is desired to measure the cell number or expansion of the cellpopulation, this may be done for example by taking an aliquot andperforming immunocytochemistry (e.g. to count nuclei stained with SytoxOrange) or by live cell imaging under brightfield microscope to countthe number of cells or by performing real-time quantitative live-cellanalysis of cell confluence at various time points during the cellpopulation expansion phase of the method according to the invention.

In one aspect according to the invention the CEC population obtainableor obtained by the method of cell population expansion according to theinvention preferably shows at least one of the followingcharacteristics. More preferably, it shows two or more, particularlypreferably all, of the following characteristics.

-   (1) The cells express Na/K ATPase. The expression of Na/K ATPase may    be estimated by standard techniques known in the art, such as for    example immunohistochemistry, quantitative RT-PCR or by FACS    analysis.-   (2) The cells express one or more of Collagen 8a2, AQP1    (aquaporin 1) and SLC4A11 (Solute Carrier Family 4 Member 11).    Preferably the relative expression levels are higher than cells    which do not typically express collagen 8a2, AQP1 and SLC4A11, such    as, for example, in dermal fibroblasts. The expression of Collagen    8a2, AQP1 or SLC4A11 may be estimated by standard techniques known    in the art, such as for example immunohistochemistry, quantitative    RT-PCR or by FACS analysis.-   (3) The cells do not express (or at most express relatively low    levels of) RPE65 (a marker of retinal pigmented epithelium) and/or    CD31 (a marker of vascular endothelium). The relative expression    levels are similar to cells which do not typically express RPE65,    CD31, such as in dermal fibroblasts. The expression of RPE65 and    CD31 may be estimated by standard techniques known in the art, such    as for example quantitative RT-PCR, immunohistochemistry or FACS    analysis.-   (4) The cells express relatively low levels of CD73. The relative    expression levels are lower than cells which have undergone    endothelial to mesenchymal transition. The expression of CD73 may be    estimated by standard techniques known in the art, such as for    example FACS analysis or immunohistochemistry.

In another aspect according to the invention, when in a layer, forexample when cultured on a plate, the CEC population obtainable by themethod of cell population expansion according to the inventionpreferably shows at least one of the following characteristics. Morepreferably, it shows two or more, particularly preferably all, of thefollowing characteristics:

-   (1) The cells are able to form a single layer structure. This is one    of the characteristics of the corneal endothelial cell layer in the    body. This may be observed by nuclear staining (e.g. with nuclear    dye such as Sytox, Hoechst) followed by examination by microscopy.-   (2) The cells are able to form tight junctions. This may be checked    by a standard technique known in the art, immunofluorescence    staining of tight-junction marker Zonula Occludens-1 (ZO-1).-   (3) The cells are able to be regularly arranged in the cell layer.    This may be checked by a standard technique known in the art,    immunofluorescence staining of tight-junction marker Zonula    Occludens-1 (ZO-1). In the healthy corneal endothelial cell layer in    the body, the cells constituting the layer are regularly arrayed,    due to which corneal endothelial cells are considered to maintain    normal function and high transparency and the cornea is considered    to appropriately exhibit water control function.

The cell population expanded by the method of cell population expansionaccording to the invention may be added to a solution and then stored,for example in a preservation or cryopreservation solution (such asthose described below), or added directly to a composition suitable forocular delivery. The preservation, cryopreservation solution orcomposition suitable for ocular delivery may optionally comprise a LATSinhibitor according to the invention.

In a more preferred embodiment according to the invention, the cellpopulation preparation which is delivered to the eye comprises very lowto negligible levels of a LATS inhibitor compound. Thus in a specificembodiment, the method of cell population expansion according to theinvention comprises the further step of rinsing to substantially removethe compound according to the invention (such as the compound accordingto Formula A1 or subformulae thereof). This may involve rinsing thecells after the cell population expansion phase according to theinvention (directly after the cell population expansion phase and/orafter the cells have been cultured to form a mature corneal endotheliumin growth medium which has not been supplemented by a LATS inhibitor).To rinse the cells, the cells are centrifuged, and a cell suspension ismade in PBS or growth medium according to the invention. This step maybe performed multiple times, e.g. one to ten times, to rinse out thecells. Finally the cells may be resuspended in a preservation solution,cryopreservation solution, a composition suitable for ocular delivery,growth medium or combinations thereof as desired.

The expanded population of cells prepared by the method of cellpopulation expansion and rinsed of cell proliferation medium comprisinga LATS inhibitor according the invention may be transferred to acomposition suitable for ocular delivery, such as for example alocalising agent. Optionally the cell population is stored for a periodbefore addition to a localising agent suitable for ocular delivery. In apreferred embodiment, the expanded cell population may first be added toa solution suitable for preservation or cryopreservation, whichpreferably does not comprise a LATS inhibitor, and the cell populationstored (optionally with freezing) before addition to a localising agentsuitable for ocular delivery, which also preferably does not comprise aLATS inhibitor.

Typical solutions for suitable for preservation of CECs are Optisol orPBS, preferably Optisol. Optisol is a corneal storage medium comprisingchondroitin sulfate and dextran to enhance corneal dehydration duringstorage (see for example Kaufman et al., (1991) Optisol corneal storagemedium; Arch Ophthalmol June; 109(6): 864-8). For cryopreservation,glycerol, dimethyl sulfoxide, propylene glycol or acetamide may be usedin the cryopreservation solution of the present invention. Thecryopreserved preparation of cells is typically kept at −20° C. or −80°C.

In one aspect the invention relates to a preserved or cryopreservedpreparation of corneal endothelial cells obtainable by the method ofcell population expansion according to the invention. In an alternativeaspect the invention relates to a fresh cell preparation where cornealendothelial cells obtainable by the method of cell population expansionaccording to the invention are in suspension in PBS and/or growth mediumor combined with a localising agent. The fresh cell preparation istypically kept at about 37° C. Standard cell cultures containers knownin the art may be used to store the cells, such as a vial or a flask.

In a preferred embodiment according to the invention, before use in theeye, a cryopreserved preparation of cells is thawed (for example byincubating at a temperature of about 37° C. in an incubator orwaterbath). Preferably 10 volumes of PBS or growth medium may be addedto rinse off the cells from the cryopreservant solution. Cells may thenbe rinsed by centrifugation, and a cell suspension may be made in PBSand/or growth medium, before combination with a localising agent forocular delivery, which also preferably does not comprise a LATSinhibitor.

In one aspect of the invention the expanded population of cells preparedby the method of cell population expansion, (preferably also includingthe step of growth in medium without supplementation with LATS inhibitorto form a mature corneal endothelium), are prepared as a suspension (forexample in PBS and/or growth medium, such as for example X-VIVO medium)and combined with a localising agent suitable for ocular delivery, (suchas a biomatrix like GelMA). In a specific embodiment of the method oftreatment according to the invention, this combination of cells, PBSand/or growth medium, and biomatrix is delivered as a suspension to theeye. In yet another specific embodiment this combination of cells, PBSand/or growth medium, and biomatrix comprises at most only trace levelsof a LATS inhibitor.

Alternatively, the cells may be cultured and the cell populationproliferation phase may occur in cell proliferation medium on alocalising agent suitable for cell delivery to the ocular surface.

In an embodiment of the invention the cell population expanded accordingto the invention may be isolated as a contiguous cell sheet for deliveryto the cornea, using methods known in the art (for examples, see Kim etal, JSM Biotechnol. Bioeng., 2016, p. 1047). Cell sheets may bemechanically supported on a material or materials for delivery to thecornea.

Reduction of Immunorejection

Upon transplantation, allogeneic limbal stem cells are at risk ofrejection by the recipient's immune system. Immunosuppression regimenscan be used to reduce the risk of immunorejection of transplanted cells,such as LSCs.

Suitable systemic immunosuppressant agents used in recipients ofallogeneic LSCs include tacrolimus, mycophenolate mofetil, prednisoneand prophylactic valganciclovir and trimethoprim/sulfamethoxazole. (See:Holland E J, Mogilishetty G, Skeens H M, Hair D B, Neff K D, Biber J M,Chan C C (2012) Systemic immunosuppression in ocular surface stem celltransplantation: results of a 10-year experience. Cornea. 2012 June;31(6):655-61).

As the methods of cell population expansion according the presentinvention provide high expansion capabilities of a population of cells,optionally gene-editing technologies may be used to remove drivers ofimmunorejection or add genes that reduce the recipient's immuneresponse.

In one aspect of the invention gene editing is carried out on a cellpopulation “ex vivo”. In another aspect of the invention gene-editingtechnologies may optionally be used to reduce or eliminate theexpression of a gene associated with facilitating a host versus graftimmune response. In a preferred embodiment the gene is selected from thegroup consisting of: B2M, HLA-A, HLA-B and HLA-C. In a specificembodiment the gene is B2M. B2M is beta 2 microglobulin and is acomponent of the class I major histocompatibility complex (MHC). It hasthe HUGO Gene Nomenclature Committee (HGNC) identifier 914. HLA-A ismajor histocompatibility complex, class I, A (HGNC ID 4931). HLA-B ismajor histocompatibility complex, class I, B (HGNC ID 4932). HLA-C ismajor histocompatibility complex, class I, C (HGNC ID 4933).

Several gene editing methods may be used, including, but not limited tomethods selected from the group consisting of CRISPR (CRISPR: clusteredregularly interspaced short palindromic repeats, also known asCRISPR/Cas systems), ZFN (Zinc-finger nucleases), TALEN (transcriptionactivator-like effector based nucleases), engineered meganucleases (e.g.ARCUS nucleases, such as the ARC nuclease), AAV vector (adeno-associatedvirus) gene delivery (e.g., AAV vector driven homologous recombination)and lentiviral vectors-based genome editing technologies. AAV vectordriven gene delivery, such as driven by homologous recombination, can beachieved by an AAV selected from the group consisting of: AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or a derivative thereof. In oneaspect of the invention gene editing is carried out on a cell population“ex vivo”.

Gene Editing System

As used herein, the term “gene editing system” refers to a systemcomprising one or more DNA-binding domains or components and one or moreDNA-modifying domains or components, or isolated nucleic acids, e.g.,one or more vectors, encoding said DNA-binding and DNA-modifying domainsor components. Gene editing systems are used for modifying the nucleicacid of a target gene and/or for modulating the expression of a targetgene. In known gene editing systems, for example, the one or moreDNA-binding domains or components are associated with the one or moreDNA-modifying domains or components, such that the one or moreDNA-binding domains target the one or more DNA-modifying domains orcomponents to a specific nucleic acid site. As described herein, geneediting can be carried out ex vivo.

AAV Gene Editing Systems

The use of vectors derived from AAVs to transfer genes in vitro and invivo is well known in the art (see U.S. Pat. No. 9,707,304, forexample). The use of viral vectors for editing genes in a cell has beendescribed, for example, in Chen et al., 2016, Molecular Therapy,24:447-457; Gornalusse et al., Nature Biotechnology, 2017,35(8):765-772; and WO2017087961. An AAV vector can be made using methodsknown in the art. Such methods are described, for example, in Flotte TR. Adeno-associated virus-based gene therapy for inherited disorders.Pediatr Res. 2005 December; 58(6):1143-7; Goncalves M A.Adeno-associated virus: from defective virus to effective vector, VirolJ. 2005 May 6; 2:43; Surace E M, Auricchio A. Adeno-associated viralvectors for retinal gene transfer. Prog Retin Eye Res. 2003 November;22(6):705-19; Mandel R J, Manfredsson F P, Foust K D, Rising A,Reimsnider S, Nash K, Burger C. Recombinant adeno-associated viralvectors as therapeutic agents to treat neurological disorders. Mol Ther.2006 March; 13(3):463-83.

CRISPR Gene Editing Systems

“CRISPR” as used herein refers to a set of clustered regularlyinterspaced short palindromic repeats, or a system comprising such a setof repeats. “Cas,” as used herein, refers to a CRISPR-associatedprotein. The diverse CRISPR-Cas systems can be divided into two classesaccording to the configuration of their effector modules: class 1 CRISPRsystems utilize several Cas proteins and the crRNA to form an effectorcomplex, whereas class 2 CRISPR systems employ a large single-componentCas protein in conjunction with crRNAs to mediate interference. Oneexample of class 2 CRISPR-Cas system employs Cpf1 (CRISPR fromPrevotella and Francisella 1). See, e.g., Zetsche et al., Cell163:759-771 (2015), the content of which is herein incorporated byreference in its entirety. The term “Cpf1” as used herein includes allorthologs, and variants that can be used in a CRISPR system.

Naturally-occurring CRISPR systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

The CRISPR system has been modified for use in gene editing (silencing,enhancing or changing specific genes) in eukaryotes such as mice,primates and humans. Wiedenheft et al. (2012) Nature 482: 331-8. This isaccomplished by, for example, introducing into the eukaryotic cell oneor more vectors encoding a specifically engineered guide RNA (gRNA)(e.g., a gRNA comprising sequence complementary to sequence of aeukaryotic genome) and one or more appropriate RNA-guided nucleases,e.g., Cas proteins. The RNA guided nuclease forms a complex with thegRNA, which is then directed to the target DNA site by hybridization ofthe gRNA's sequence to complementary sequence of a eukaryotic genome,where the RNA-guided nuclease then induces a double or single-strandbreak in the DNA. Insertion or deletion of nucleotides at or near thestrand break creates the modified genome.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs.

A simpler CRISPR system relies on the protein Cas9, which is a nucleasewith two active cutting sites, one for each strand of the double helix.Combining Cas9 and modified CRISPR locus RNA can be used in a system forgene editing. Pennisi (2013) Science 341: 833-836.

In some embodiments, the RNA-guided nuclease is a Cas molecule, e.g., aCas9 molecule. The “Cas9 molecule,” can interact with a gRNA molecule(e.g., sequence of a domain of a tracr, also known as tracrRNA or transactivating CRISPR RNA) and, in concert with the gRNA molecule, localize(e.g., target or home) to a site which comprises a target sequence andPAM (protospacer adjacent motif) sequence.

According to the present invention, Cas9 molecules of, derived from, orbased on the Cas9 proteins of a variety of species can be used in themethods and compositions described herein. For example, Cas9 moleculesof, derived from, or based on, e.g., S. pyogenes, S. thermophilus,Staphylococcus aureus and/or Neisseria meningitidis Cas9 molecules, canbe used in the systems, methods and compositions described herein.Additional Cas9 species include: Acidovorax avenae, Actinobacilluspleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis,Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans,Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroidessp., Blastopirellula marina, Bradyrhiz'obium sp., Brevibacilluslatemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacterlad, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridiumperfringens, Corynebacterium accolens, Corynebacterium diphtheria,Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacteriumdolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus,Haemophilus parainfluenzae, Haemophilus sputorum, Helicobactercanadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobaclerpolytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii,Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp.,Methylosinus trichosporium, Mobiluncus mulieris, Neisseriabacilliformis, Neisseria cinerea, Neisseria flavescens, Neisserialactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp.,Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp.,Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcussp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., orVerminephrobacter eiseniae.

In some embodiments, the ability of an active Cas9 molecule to interactwith and cleave a target nucleic acid is PAM sequence dependent. A PAM(protospacer adjacent motif) sequence is a sequence in the targetnucleic acid. It is typically short, for example 2 to 7 base pairs long.In an embodiment, cleavage of the target nucleic acid occurs upstreamfrom the PAM sequence. Active Cas9 molecules from different bacterialspecies can recognize different sequence motifs (e.g., PAM sequences).In an embodiment, an active Cas9 molecule of S. pyogenes recognizes thesequence motif NGG and directs cleavage of a target nucleic acidsequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.See, e.g., Mali el al, SCIENCE 2013; 339(6121): 823-826. In anembodiment, an active Cas9 molecule of S. thermophilus recognizes thesequence motif NGGNG (SEQ ID NO: 4) and NNAG AAW (SEQ ID NO: 5) (W=A orT and N is any nucleobase) and directs cleavage of a core target nucleicacid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from thesesequences. See, e.g., Horvath et al., SCIENCE 2010; 327(5962): 167-170,and Deveau et al, J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment,an active Cas9 molecule of S. mutans recognizes the sequence motif NGGor NAAR (R-A or G) and directs cleavage of a core target nucleic acidsequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence.See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400.

In an embodiment, an active Cas9 molecule of S. aureus recognizes thesequence motif NNGRR (SEQ ID NO: 6) (R=A or G) and directs cleavage of atarget nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstreamfrom that sequence. See, e.g., Ran F. et al., NATURE, vol. 520, 2015,pp. 186-191. In an embodiment, an active Cas9 molecule of N.meningitidis recognizes the sequence motif NNNNGATT (SEQ ID NO: 7) anddirects cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to5, base pairs upstream from that sequence. See, e.g., Hou et al., PNASEARLY EDITION 2013, 1-6. The ability of a Cas9 molecule to recognize aPAM sequence can be determined, e.g., using a transformation assaydescribed in Jinek et al, SCIENCE 2012, 337:816.

Exemplary naturally occurring Cas9 molecules are described in Chylinskiet al, RNA Biology 2013; 10:5, 727-737. Such Cas9 molecules include Cas9molecules of a cluster 1 bacterial family, cluster 2 bacterial family,cluster 3 bacterial family, cluster 4 bacterial family, cluster 5bacterial family, cluster 6 bacterial family, a cluster 7 bacterialfamily, a cluster 8 bacterial family, a cluster 9 bacterial family, acluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12bacterial family, a cluster 13 bacterial family, a cluster 14 bacterialfamily, a cluster 15 bacterial family, a cluster 16 bacterial family, acluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19bacterial family, a cluster 20 bacterial family, a cluster 21 bacterialfamily, a cluster 22 bacterial family, a cluster 23 bacterial family, acluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26bacterial family, a cluster 27 bacterial family, a cluster 28 bacterialfamily, a cluster 29 bacterial family, a cluster 30 bacterial family, acluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33bacterial family, a cluster 34 bacterial family, a cluster 35 bacterialfamily, a cluster 36 bacterial family, a cluster 37 bacterial family, acluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40bacterial family, a cluster 41 bacterial family, a cluster 42 bacterialfamily, a cluster 43 bacterial family, a cluster 44 bacterial family, acluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47bacterial family, a cluster 48 bacterial family, a cluster 49 bacterialfamily, a cluster 50 bacterial family, a cluster 51 bacterial family, acluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54bacterial family, a cluster 55 bacterial family, a cluster 56 bacterialfamily, a cluster 57 bacterial family, a cluster 58 bacterial family, acluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61bacterial family, a cluster 62 bacterial family, a cluster 63 bacterialfamily, a cluster 64 bacterial family, a cluster 65 bacterial family, acluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68bacterial family, a cluster 69 bacterial family, a cluster 70 bacterialfamily, a cluster 71 bacterial family, a cluster 72 bacterial family, acluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75bacterial family, a cluster 76 bacterial family, a cluster 77 bacterialfamily, or a cluster 78 bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule ofa cluster 1 bacterial family. Examples include a Cas9 molecule of: S.pyogenes (e.g., strain SF370, MGAS 10270, MGAS 10750, MGAS2096, MGAS315,MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g.,strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans(e.g., strain UA 159, NN2025), S. macacae (e.g., strain NCTC1 1558), S.gallolylicus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g.,strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S.bovis (e.g., strain ATCC 700338), S. cmginosus (e.g.; strain F0211), S.agalactia* (e.g., strain NEM316, A909), Listeria monocytogenes (e.g.,strain F6854), Listeria innocua (L. innocua, e.g., strain Clip 11262),EtUerococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium(e.g., strain 1,23,408). Additional exemplary Cas9 molecules are a Cas9molecule of Neisseria meningitidis (Hou et al. PNAS Early Edition 2013,1-6) and a S. aureus Cas9 molecule.

In an embodiment, a Cas9 molecule, e.g., an active Cas9 moleculecomprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% homology with; differs at no more than1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues whencompared with; differs by at least 1, 2, 5, 10 or 20 amino acids but byno more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or isidentical to; any Cas9 molecule sequence described herein or a naturallyoccurring Cas9 molecule sequence, e.g., a Cas9 molecule from a specieslisted herein or described in Chylinski et al., RNA Biology 2013, 10:5,12I-T, 1 Hou et al. PNAS Early Edition 2013, 1-6

In an embodiment, a Cas9 molecule comprises an amino acid sequencehaving 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%homology with; differs at no more than 1%, 2%, 5%, 10%, 15%, 20%, 30%,or 40% of the amino acid residues when compared with; differs by atleast 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60,50, 40 or 30 amino acids from; or is identical to; S. pyogenes Cas9(UniProt Q99ZW2). In embodiments, the Cas9 molecule is a S. pyogenesCas9 variant, such as a variant described in Slaymaker et al., ScienceExpress, available online Dec. 1, 2015 at Science DOI:10.1126/science.aad5227; Kleinstiver et al., Nature, 529, 2016, pp.490-495, available online Jan. 6, 2016 at doi:10.1038/nature16526; orUS2016/0102324, the contents of which are incorporated herein in theirentirety.

In some embodiments, the Cas9 molecule, e.g., a Cas9 of S. pyogenes, mayadditionally comprise one or more amino acid sequences that conferadditional activity. In some aspects, the Cas9 molecule may comprise oneor more nuclear localization sequences (NLSs), such as at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. Typically, an NLS consists of one ormore short sequences of positively charged lysines or arginines exposedon the protein surface, but other types of NLS are known. Non-limitingexamples of NLSs include an NLS sequence comprising or derived from: theNLS of the SV40 virus large T-antigen, having the amino acid sequencePKKKRKV (SEQ ID NO: 8). Other suitable NLS sequences are known in theart (e.g., Sorokin, Biochemistry (Moscow) (2007) 72:13, 1439-1457; LangeJ Biol Chem. (2007) 282:8, 5101-5). In any of the aforementionedembodiments, the Cas9 molecule may additionally (or alternatively)comprise a tag, e.g., a His tag, e.g., a His(6) tag (SEQ ID NO: 25) orHis(8) tag (SEQ ID NO: 26), e.g., at the N terminus or the C terminus.

Thus, engineered CRISPR gene editing systems, e.g., for gene editing ineukaryotic cells, typically involve (1) a guide RNA molecule (gRNA)comprising a targeting domain (which is capable of hybridizing to thegenomic DNA target sequence), and a sequence which is capable of bindingto a Cas, e.g., Cas9 enzyme, and (2) a Cas, e.g., Cas9, protein. Thesequence which is capable of binding to a Cas protein may comprise adomain referred to as a tracr domain or tracrRNA. The targeting domainand the sequence which is capable of binding to a Cas, e.g., Cas9enzyme, may be disposed on the same (sometimes referred to as a singlegRNA, chimeric gRNA or sgRNA) or different molecules (sometimes referredto as a dual gRNA or dgRNA). If disposed on different molecules, eachincludes a hybridization domain which allows the molecules to associate,e.g., through hybridization. gRNA molecule formats are known in the art.An exemplary gRNA molecule, e.g., dgRNA molecule, as disclosed hereincomprises, e.g., consists of, a first nucleic acid having the sequence:

5′nnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG 3′ (SEQ ID NO: 9), wherethe “n” 's refer to the residues of the targeting domain, e.g., asdescribed herein, and may consist of 15-25 nucleotides, e.g., consistsof 20 nucleotides;

and a second nucleic acid sequence having the exemplary sequence:

5′AACUUACCAAGGAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC 3′, optionally with 1, 2, 3, 4, 5, 6, or 7(e.g., 4 or 7, e.g., 7) additional U nucleotides at the 3′ end (SEQ IDNO: 10).

The second nucleic acid molecule may alternatively consist of a fragmentof the sequence above, wherein such fragment is capable of hybridizingto the first nucleic acid. An example of such second nucleic acidmolecule is:

5′AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGC3′, optionally with 1, 2, 3, 4, 5, 6, or 7 (e.g., 4 or 7, e.g., 7)additional U nucleotides at the 3′ end (SEQ ID NO:11).

Another exemplary gRNA molecule, e.g., a sgRNA molecule, as disclosedherein comprises, e.g., consists of a first nucleic acid having thesequence:

′nnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC 3′(SEQ ID NO:12), where the “n” 'srefer to the residues of the targeting domain, e.g., as describedherein, and may consist of 15-25 nucleotides, e.g., consist of 20nucleotides, optionally with 1, 2, 3, 4, 5, 6, or 7 (e.g., 4 or 7, e.g.,4) additional U nucleotides at the 3′ end.

Additional components and/or elements of CRISPR gene editing systemsknown in the art, e.g., are described in U.S. Publication No.2014/0068797, WO2015/048577, and Cong (2013) Science 339: 819-823, thecontents of which are hereby incorporated by reference in theirentirety. Such systems can be generated which inhibit a target gene, by,for example, engineering a CRISPR gene editing system to include a gRNAmolecule comprising a targeting domain that hybridizes to a sequence ofthe target gene. In embodiments, the gRNA comprises a targeting domainwhich is fully complementarity to 15-25 nucleotides, e.g., 20nucleotides, of a target gene. In embodiments, the 15-25 nucleotides,e.g., 20 nucleotides, of the target gene, are disposed immediately 5′ toa protospacer adjacent motif (PAM) sequence recognized by the RNA-guidednuclease, e.g., Cas protein, of the CRISPR gene editing system (e.g.,where the system comprises a S. pyogenes Cas9 protein, the PAM sequencecomprises NGG, where N can be any of A, T, G or C).

In some embodiments, the gRNA molecule and RNA-guided nuclease, e.g.,Cas protein, of the CRISPR gene editing system can be complexed to forma RNP (ribonucleoprotein) complex. Such RNP complexes may be used in themethods described herein. In other embodiments, nucleic acid encodingone or more components of the CRISPR gene editing system may be used inthe methods described herein.

In some embodiments, foreign DNA can be introduced into the cell alongwith the CRISPR gene editing system, e.g., DNA encoding a desiredtransgene, with or without a promoter active in the target cell type.Depending on the sequences of the foreign DNA and target sequence of thegenome, this process can be used to integrate the foreign DNA into thegenome, at or near the site targeted by the CRISPR gene editing system.For example, 3′ and 5′ sequences flanking the transgene may be includedin the foreign DNA which are homologous to the gene sequence 3′ and 5′(respectively) of the site in the genome cut by the gene editing system.Such foreign DNA molecule can be referred to “template DNA.”

In an embodiment, the CRISPR gene editing system of the presentinvention comprises Cas9, e.g., S. pyogenes Cas9, and a gRNA comprisinga targeting domain which hybridizes to a sequence of a gene of interest.In an embodiment, the gRNA and Cas9 are complexed to form aRNP(ribonucleoprotein). In an embodiment, the CRISPR gene editing systemcomprises nucleic acid encoding a gRNA and nucleic acid encoding a Casprotein, e.g., Cas9, e.g., S. pyogenes Cas9. In an embodiment, theCRISPR gene editing system comprises a gRNA and nucleic acid encoding aCas protein, e.g., Cas9, e.g., S. pyogenes Cas9.

In some embodiments, inducible control over Cas9, sgRNA expression canbe utilized to optimize efficiency while reducing the frequency ofoff-target effects thereby increasing safety. Examples include, but arenot limited to, transcriptional and post-transcriptional switches listedas follows; doxycycline inducible transcription Loew et al. (2010) BMCBiotechnol. 10:81, Shield1 inducible protein stabilization Banaszynskiet al. (2016) Cell 126: 995-1004, Tamoxifen induced protein activationDavis et al. (2015) Nat. Chem. Biol. 11: 316-318, Rapamycin oroptogenetic induced activation or dimerization of split Cas9 Zetsche(2015) Nature Biotechnol. 33(2): 139-142, Nihongaki et al. (2015) NatureBiotechnol. 33(7): 755-760, Polstein and Gersbach (2015) Nat. Chem.Biol. 11: 198-200, and SMASh tag drug inducible degradation Chung et al.(2015) Nat. Chem. Biol. 11: 713-720.

In general, the CRISPR-Cas or CRISPR system refers collectively totranscripts and other elements involved in the expression of ordirecting the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, a tracr (trans-activating CRISPR)sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-matesequence (encompassing a “direct repeat” and a tracrRNA-processedpartial direct repeat in the context of an endogenous CRISPR system), aguide sequence (also referred to as a “spacer” in the context of anendogenous CRISPR system), or “RNA(s)” as that term is herein used(e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr)RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences andtranscripts from a CRISPR locus. In general, a CRISPR system ischaracterized by elements that promote the formation of a CRISPR complexat the site of a target sequence (also referred to as a protospacer inthe context of an endogenous CRISPR system). In the context of formationof a CRISPR complex, “target sequence” refers to a sequence to which aguide sequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide sequence promotes the formation ofa CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In someembodiments it may be preferred in a CRISPR complex that the tracrsequence has one or more hairpins and is 30 or more nucleotides inlength, 40 or more nucleotides in length, or 50 or more nucleotides inlength; the guide sequence is between 10 to 30 nucleotides in length,the CRISPR/Cas enzyme is a Type II Cas9 enzyme. In embodiments of theinvention the terms guide sequence and guide RNA are usedinterchangeably. In general, a guide sequence is any polynucleotidesequence having sufficient complementarity with a target polynucleotidesequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies); ELAND (Illumina,San Diego, Calif.), and SOAP. In some embodiments, a guide sequence isabout or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or morenucleotides in length. In some embodiments, a guide sequence is lessthan about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotidesin length. Preferably the guide sequence is 10-30 nucleotides long. Theability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome. For example, for the S. pyogenes Cas9, a uniquetarget sequence in a genome may include a Cas9 target site of the formMM M MMMNNNNNNNNNNNNXGG (SEQ ID NO: 13), where NNN NNN NN XGG (N is A,G, T, or C; and X can be anything) has a single occurrence in thegenome. A unique target sequence in a genome may include an S. pyogenesCas9 target site of the form MMM MMMMMNNNNNNNNNNNXGG (SEQ ID NO: 14),where N N N N XGG (N is A, G, T, or C; and X can be anything) has asingle occurrence in the genome. For the S. thermophilus CRISPRI Cas9, aunique target sequence in a genome may include a Cas9 target site of theform MMMMMMMMNN N N NN XXAGAAW (SEQ ID NO: 15), where NNN NN N XXAGAAW(SEQ ID NO: 29) (N is A, G, T, or C; X can be anything; and W is A or T)has a single occurrence in the genome. A unique target sequence in agenome may include an S. thermophilus CRISPRI Cas9 target site of theform MMMMMM MN N NNN NNXXAGAAW (SEQ ID NO: 16), where NNNNNNNNNNNXXAGAAW(SEQ ID NO: 30) (N is A, G, T, or C; X can be anything; and W is A or T)has a single occurrence in the genome. For the S. pyogenes Cas9, aunique target sequence in a genome may include a Cas9 target site of theform MMMMMMMMNNNN NNNNNNXGGXG (SEQ ID NO: 17), where NNNNNNNNNNNNXGGXG(N is A, G, T, or C; and X can be anything) has a single occurrence inthe genome. A unique target sequence in a genome may include an S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ IDNO: 31) where NNNNNNNNNNNXGGXG (SEQ ID NO: 18), (N is A, G, T, or C; andX can be anything) has a single occurrence in the genome. In each ofthese sequences, N is any nucleobase and “M” may be A, G, T, or C, andneed not be considered in identifying a sequence as unique. In someembodiments, a guide sequence is selected to reduce the degree secondarystructure within the guide sequence. In some embodiments, about or lessthan about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer ofthe nucleotides of the guide sequence participate in self-complementarybase pairing when optimally folded. Optimal folding may be determined byany suitable polynucleotide folding algorithm. Some programs are basedon calculating the minimal Gibbs free energy. An example of one suchalgorithm is mFold, as described by Zuker and Stiegler (Nucleic AcidsRes. 9 (1981), 133-148). Another example folding algorithm is the onlinewebserver RNAfold, developed at Institute for Theoretical Chemistry atthe University of Vienna, using the centroid structure predictionalgorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; andPA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1 151-62).

Methods for Designing gRNA Molecules

Methods for selecting, designing, and validating targeting domains foruse in the gRNAs described herein are provided. Exemplary targetingdomains for incorporation into gRNAs are also provided herein.

Methods for selection and validation of target sequences as well asoff-target analyses have been described (see, e.g., Mali 2013; Hsu 2013;Fu 2014; Heigwer 2014; Bae 2014; and Xiao 2014). For example, targetsequences can be chosen by identifying the PAM sequence for a Cas9molecule (for example, relevant PAM e.g., NGG PAM for S. pyogenes,NNNNGATT (SEQ ID NO: 19), or NNNNGCTT PAM (SEQ ID NO: 20), for N.meningitides, and NNGRRT (SEQ ID NO: 21), or NNGRRV PAM (SEQ ID NO: 22),for S. aureus), and identifying the adjacent sequence as the targetsequence for a CRISPR system using that Cas9 molecule. A software toolcan be used to further refine the choice of potential targeting domainscorresponding to a user's target sequence, e.g., to minimize totaloff-target activity across the genome. Candidate targeting domains andgRNAs comprising those targeting domains can be functionally evaluatedby using methods known in the art and/or as set forth herein.

As a non-limiting example, targeting domains for use in gRNAs for usewith S. pyogenes, N. meningiitidis and S. aureus Cas9s are identifiedusing a DNA sequence searching algorithm. 17-mer, 18-mer, 19-mer,20-mer, 21-mer, 22-mer, 23-mer, and/or 24-mer targeting domains aredesigned for each Cas9. With respect to S. pyogenes Cas9, preferably,the targeting domain is a 20-mer. gRNA design is carried out using acustom gRNA design software based on the public tool cas-offinder (Bae2014). This software scores guides after calculating their genome-wideoff-target propensity.

Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9molecule/gRNA molecule complexes, can be evaluated by art-known methodsor as described herein. For example, exemplary methods for evaluatingthe endonuclease activity of Cas9 molecule have been describedpreviously (Jinek 2012). Each technique described herein may be usedalone or in combination with one or more techniques to evaluate thecandidate molecule. The techniques disclosed herein may be used for avariety of methods including, without limitation, methods of determiningthe stability of a Cas9 molecule/gRNA molecule complex, methods ofdetermining a condition that promotes a stable Cas9 molecule/gRNAmolecule complex, methods of screening for a stable Cas9 molecule/gRNAmolecule complex, methods of identifying an optimal gRNA to form astable Cas9 molecule/gRNA molecule complex, and methods of selecting aCas9/gRNA complex for administration to a subject.

Binding and Cleavage Assay: Testing the endonuclease activity of Cas9molecule The ability of a Cas9 molecule/gRNA molecule complex to bind toand cleave a target nucleic acid can be evaluated in a plasmid cleavageassay. In this assay, synthetic or in vitro-transcribed gRNA molecule ispre-annealed prior to the reaction by heating to 95° C. and slowlycooling down to room temperature. Native or restrictiondigest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 minat 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA(50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5,150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl2. Thereactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS,250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. The resulting cleavage productsindicate whether the Cas9 molecule cleaves both DNA strands, or only oneof the two strands. For example, linear DNA products indicate thecleavage of both DNA strands. Nicked open circular products indicatethat only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 molecule/gRNA molecule complex tobind to and cleave a target nucleic acid can be evaluated in anoligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides(10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotidekinase and −3-6 pmol (−20-40 mCi) [y-32P]-ATP in IX T4 polynucleotidekinase reaction buffer at 37° C. for 30 min, in a 50 microlitrereaction. After heat inactivation (65° C. for 20 min), reactions arepurified through a column to remove unincorporated label. Duplexlocalising agents (100 nM) are generated by annealing labeledoligonucleotides with equimolar amounts of unlabeled complementaryoligonucleotide at 95° C. for 3 min, followed by slow cooling to roomtemperature. For cleavage assays, gRNA molecules are annealed by heatingto 95° C. for 30 s, followed by slow cooling to room temperature. Cas9(500 nM final concentration) is pre-incubated with the annealed gRNAmolecules (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mMKCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in a total volume of 9microlitre. Reactions are initiated by the addition of 1 microlitretarget DNA (10 nM) and incubated for 1 h at 37° C. Reactions arequenched by the addition of 20 microlitre of loading dye (5 mM EDTA,0.025% SDS, 5% glycerol in formamide) and heated to 95° C. for 5 min.Cleavage products are resolved on 12% denaturing polyacrylamide gelscontaining 7 M urea and visualized by phosphorimaging. The resultingcleavage products indicate that whether the complementary strand, thenon-complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of acandidate gRNA molecule or candidate Cas9 molecule.

Indel Detection and Identification. Targeted genome modifications canalso be detected by either Sanger or deep sequencing. For the former,genomic DNA from the modified region can be amplified with eitherprimers flanking the target sequence of the gRNA. Amplicons can besubcloned into a plasmid such as pUC19 for transformation, andindividual colonies should be sequenced to reveal the clonal genotype.

Alternatively, deep sequencing is suitable for sampling a large numberof samples or target sites. NGS primers are designed for shorteramplicons, typically in the 100-200-bp size range. For the detection ofindels, it is important to design primers situated at least 50 bp fromthe Cas9 target site to allow for the detection of longer indels.Amplicons may be assessed using commercially-available instruments, forexample, the Illumina system. Detailed descriptions of NGS optimizationand troubleshooting can be found in the Illumina user manual.

TALEN Gene Editing Systems

TALENs are produced artificially by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. Transcription activator-like effects(TALEs) can be engineered to bind any desired DNA sequence, e.g., atarget gene. By combining an engineered TALE with a DNA cleavage domain,a restriction enzyme can be produced which is specific to any desiredDNA sequence. These can then be introduced into a cell, wherein they canbe used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; andBoch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA bindingdomain contains a repeated, highly conserved 33-34 amino acid sequence,with the exception of the 12th and 13th amino acids. These two positionsare highly variable, showing a strong correlation with specificnucleotide recognition. They can thus be engineered to bind to a desiredDNA sequence.

To produce a TALEN, a TALE protein is fused to a nuclease (N), which is,for example, a wild-type or mutated Fokl endonuclease. Several mutationsto Fokl have been made for its use in TALENs; these, for example,improve cleavage specificity or activity. Cermak et al. (2011) NucI.Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8;Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011)Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepeket al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol.Biol. 200: 96.

The Fokl domain functions as a dimer, requiring two constructs withunique DNA binding domains for sites in the target genome with properorientation and spacing. Both the number of amino acid residues betweenthe TALE DNA binding domain and the Fokl cleavage domain and the numberof bases between the two individual TALEN binding sites appear to beimportant parameters for achieving high levels of activity. Miller etal. (2011) Nature Biotech. 29: 143-8.

A TALEN (or pair of TALENs) can be used inside a cell to produce adouble-stranded break (DSB). A mutation can be introduced at the breaksite if the repair mechanisms improperly repair the break vianon-homologous end joining. For example, improper repair may introduce aframe shift mutation. Alternatively, foreign DNA can be introduced intothe cell along with the TALEN, e.g., DNA encoding a transgene, anddepending on the sequences of the foreign DNA and chromosomal sequence,this process can be used to integrate the transgene at or near the sitetargeted by the TALEN. TALENs specific to a target gene can beconstructed using any method known in the art, including various schemesusing modular components. Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6: e19509; U.S. Pat. Nos.8,420,782; 8,470,973, the contents of which are hereby incorporated byreference in their entirety.

Zinc Finger Nuclease (ZFN) Gene Editing System

“ZFN” or “Zinc Finger Nuclease” refer to a zinc finger nuclease, anartificial nuclease which can be used to modify, e.g., delete one ormore nucleic acids of, a desired nucleic acid sequence.

Like a TALEN, a ZFN comprises a Fokl nuclease domain (or derivativethereof) fused to a DNA-binding domain. In the case of a ZFN, theDNA-binding domain comprises one or more zinc fingers. Carroll et al.(2011) Genetics Society of America 188: 773-782; and Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93: 1156-1160.

A zinc finger is a small protein structural motif stabilized by one ormore zinc ions. A zinc finger can comprise, for example, Cys2His2, andcan recognize an approximately 3-bp sequence. Various zinc fingers ofknown specificity can be combined to produce multi-finger polypeptideswhich recognize about 6, 9, 12, 15 or 18-bp sequences. Various selectionand modular assembly techniques are available to generate zinc fingers(and combinations thereof) recognizing specific sequences, includingphage display, yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNsare required to target non-palindromic DNA sites. The two individualZFNs must bind opposite strands of the DNA with their nucleases properlyspaced apart. Bitinaite et al. (1998) Proc. Nat. Acad. 20 Sci. USA 95:10570-5.

Also like a TALEN, a ZFN can create a double-stranded break in the DNA,which can create a frame-shift mutation if improperly repaired, leadingto a decrease in the expression and amount of the target gene in a cell.ZFNs can also be used with homologous recombination to mutate the targetgene or locus, or to introduce nucleic acid encoding a desired transgeneat a site at or near the targeted sequence.

ZFNs specific to sequences in a target gene can be constructed using anymethod known in the art. See, e.g., Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008)Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; U.S.Patent Publication 2011/0158957; and U.S. Patent Publication2012/0060230, the contents of which are hereby incorporated by referencein their entirety. In embodiments, The ZFN gene editing system may alsocomprise nucleic acid encoding one or more components of the ZFN geneediting system.

Meganuclease Gene Editing System

“Meganuclease” refers to a meganuclease, an artificial nuclease whichcan be used to edit a target gene.

Meganucleases are derived from a group of nucleases which recognize15-40 base-pair cleavage sites. Meganucleases are grouped into familiesbased on their structural motifs which affect nuclease activity and/orDNA recognition. Members of the LAGLIDADG (SEQ ID NO: 23) family arecharacterized by having either one or two copies of the conservedLAGLIDADG motif (SEQ ID NO: 23) (see Chevalier et al. (2001), NucleicAcids Res. 29(18): 3757-3774). The LAGLIDADG (SEQ ID NO: 23)meganucleases with a single copy of the LAGLIDADGmotif (SEQ ID NO: 23)form homodimers, whereas members with two copies of the LAGLIDADG motif(SEQ ID NO: 23) are found as monomers. The GIY-YIG family members have aGIY-YIG module, which is 70-100 residues long and includes four or fiveconserved sequence motifs with four invariant residues, two of which arerequired for activity (see Van Roey et al. (2002), Nature Struct. Biol.9: 806-811). The His-Cys box meganucleases are characterized by a highlyconserved series of histidines and cysteines over a region encompassingseveral hundred amino acid residues (see Chevalier et al. (2001),Nucleic Acids Res. 29(18): 3757-3774). The NHN family, the members aredefined by motifs containing two pairs of conserved histidinessurrounded by asparagine residues (see Chevalier et al. (2001), NucleicAcids Res. 29(18): 3757-3774).

Strategies for engineering a meganuclease with altered DNA-bindingspecificity, e.g., to bind to a predetermined nucleic acid sequence areknown in the art. E.g., Chevalier et al. (2002), Mol. Cell., 10:895-905;Epinat et al. (2003) Nucleic Acids Res 31: 2952-62; Silva et al. (2006)J Mol Biol 361: 744-54; Seligman et al. (2002) Nucleic Acids Res 30:3870-9; Sussman et al. (2004) J Mol Biol 342: 31-41; Rosen et al. (2006)Nucleic Acids Res; Doyon et al. (2006) J. Am Chem Soc 128: 2477-84; Chenet al. (2009) Protein Eng Des Sel 22: 249-56; Arnould S (2006) J MolBiol. 355: 443-58; Smith (2006) Nucleic Acids Res. 363(2): 283-94.

A meganuclease can create a double-stranded break in the DNA, which cancreate a frame-shift mutation if improperly repaired, e.g., vianon-homologous end joining, leading to a decrease in the expression of atarget gene in a cell. Alternatively, foreign DNA can be introduced intothe cell along with the Meganuclease; depending on the sequences of theforeign DNA and chromosomal sequence, this process can be used to modifya target gene, e.g., correct a defect in the target gene, thus causingexpression of a repaired target gene, or e.g., introduce such a defectinto a wt gene, thus decreasing expression of a target gene, e.g., asdescribed in Silva et al. (2011) Current Gene Therapy 11:11-27.

Ocular Administration of the Expanded Cell Population

In one aspect of the invention the expanded cell population obtainableby the methods according to the invention as described above isdelivered to the eye. The delivery is performed under asepticconditions.

In one embodiment relating to use for limbal stem cell therapy after a360° limbal peritomy the fibrovascular corneal pannus may be carefullyremoved from the surface.

In one aspect of the invention, the cell population is combined with alocalising agent suitable for ocular delivery (as described furtherbelow) and delivered to the eye. In a preferred embodiment the cells andlocalising agent suitable for ocular delivery are combined andadministered to the eye via a carrier such as for example a therapeuticcontact lens or amniotic membrane. In an alternative embodiment thecells and localising agent suitable for use in the eye, such as a lightcurable biomatrix, like GelMA, are delivered to the eye via bioprinting.

In one embodiment, the invention provides a method of transplanting apopulation of cells comprising corneal endothelial cells onto the corneaof a subject, the method comprising expanding a population of cellscomprising corneal endothelial cells by culturing said population withcell proliferation medium comprising a LATS inhibitor according to theinvention, rinsing the expanded population of cells to substantiallyremove the LATS inhibitor, and administering said cells onto the corneaof said subject. Preferably said cells are combined with a biomatrixprior to said administration. In a specific embodiment said cells arecombined with a biomatrix which is GelMA prior to said administration.In a more specific embodiment said corneal endothelial cells arecombined with a biomatrix which is bioprinted onto the ocular surface.Particularly preferably said corneal endothelial cells are combined witha biomatrix which is GelMA and bioprinted onto the ocular surface bypolymerising the GelMA by a light triggered reaction.

In another embodiment, the invention provides a method of transplantinga population of cells to the eye of a subject, comprising combining thecells with a biomatrix to form a cell/biomatrix mixture, injecting themixture into the eye of the subject or applying the mixture onto thesurface of the eye of the subject, and bioprinting the cells in or onthe eye by guiding and fixing the cells, such as on the cornea, using alight source, such as an Ultraviolet A or white light source. In certainembodiments, the light source produces light of a wavelength that is atleast 350 nm. In certain embodiments, the light source produces light inthe 350 nm to 420 nm range. For example, an LED light source can be usedto produce a light having a wavelength of 365 nm or 405 nm, or any otherwavelength above 350 nm, or a mercury lamp with a bandpass filter can beused to produce a light having a wavelength of 365 nm. In anotherembodiment, the light source produces visible, white light having awavelength, for example, in the 400 nm to 700 nm range. In certainembodiments, the cells are ocular cells, such as corneal cells (e.g.,corneal endothelial cells), lens cells, trabecular mesh cells, or cellsfound in the anterior chamber. In a particular embodiment, the cells arecorneal endothelial cells. Certain embodiments of such method include:

Embodiment x1. A method of transplanting a population of isolated cellsto the eye of a subject, comprising combining the cells with a biomatrixto form a cell/biomatrix mixture, injecting the mixture into the eye ofthe subject, (e.g., into the anterior chamber) and bioprinting the cellsin the eye by guiding and fixing the cells in the eye using a lightsource.

Embodiment x2. The method of Embodiment x1, wherein the isolated cellsare combined with a biomatrix which is GelMA and bioprinted onto thecornea by polymerising the GelMA by a light triggered reaction.

Embodiment x3. The method of Embodiment x1 or Embodiment x2, wherein thelight source produces a light having a wavelength in the 350 nm to 700nm range.

Embodiment x4. The method of any one of Embodiments x1 to x3, whereinthe wavelength is 350 nm to 420 nm.

Embodiment x5. The method of any one of Embodiments x1 to x4, whereinthe wavelength is 365 nm.

Embodiment x6. The method of any one of Embodiments x1 to x5, whereinthe isolated cells are corneal endothelial cells.

Embodiment x7. A method of transplanting a population of isolated cellsto the eye of a subject, comprising combining the cells with a biomatrixto form a cell/biomatrix mixture, applying the mixture onto the eye ofthe subject, and bioprinting the cells on the eye by guiding and fixingthe cells on the eye using a light source.

Embodiment x8. The method of Embodiment x7, wherein the isolated cellsare combined with a biomatrix which is GelMA and bioprinted onto theocular surface by polymerising the GelMA by a light triggered reaction.

Embodiment x9. The method of Embodiment x7 or Embodiment x8, wherein thelight source produces a light having a wavelength in the 350 nm to 700nm range.

Embodiment x10. The method of any one of Embodiments x7 to x9, whereinthe wavelength is 350 nm to 420 nm.

Embodiment x11. The method of any one of Embodiments x7 to x10, whereinthe wavelength is 365 nm.

Embodiment x12. The method of any one of Embodiments x7 to x11, whereinthe isolated cells are limbal stem cells.

In an alternative embodiment the expanded cell population obtainable bythe methods according to the invention as described above may bedelivered directly via a therapeutic contact lens to the eye, withoutuse of a localising agent suitable for ocular delivery (such as GelMA).

Localising Agent Suitable for Ocular Delivery

In an embodiment of the invention the cell preparation may be deliveredto the eye via a localising agent suitable for ocular use. The cells maybe embedded within the localising agent or adhered to the surface of thelocalising agent, or both.

The type of localising agent is not limited as long as it is able tocarry LSCs or CECs and is suitable for use in the eye. In a preferredembodiment, the localising agent is degradable and biocompatible. WhereCECs are delivered, preferably the localising agent can facilitate CECattachment to the cornea after surgical delivery to the surface of theeye.

In a preferred embodiment the cells are only combined with thelocalising agent after cell population expansion. In a particularlypreferred embodiment the expanded cell population is combined with thelocalising agent suitable for ocular delivery after rinsing the cellpopulation to substantially remove the presence of the LATs inhibitoraccording to the invention. In one embodiment, the LSCs or CECs andlocalising agent are combined and stored in a form suitable for ocularuse. In another embodiment, the LSCs or CECs and localising agent arestored separately and combined immediately prior to ocular use.

The localising agent is preferably selected from the list consisting offibrin, collagen, gelatin, cellulose, amniotic membrane, fibrin glue,polyethylene (glycol) diacrylate (PEGDA), GelMA, (which ismethacrylamide modified gelatin, and is also known as gelatinmethacrylate), localising agents comprising a polymer, cross-linkedpolymer, or hydrogel comprising one or more of hyaluronic acid,polyethylene glycol, polypropylene glycol, polyethylene oxide,polypropylene oxide, poloxamer, polyvinyl alcohol, polyacrylic acid,polymethacrylic acid, polyvinyl pyrrolidone, poly(lactide-co-glycolide),alginate, gelatin, collagen, fibrinogen, cellulose, methylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropylmethylcellulose, hydroxypropyl-guar, gellan gum, guar gum,xanthan gum and carboxymethylcellulose, as well as derivatives thereof,co-polymers thereof, and combinations thereof.

In a more preferred embodiment the localising agent is selected from thelist consisting of fibrin, collagen, gelatin, amniotic membrane, fibringlue, polyethylene (glycol) diacrylate (PEGDA), GelMA, localising agentscomprising a polymer, cross-linked polymer, or hydrogel comprising oneor more of hyaluronic acid, polyethylene glycol, polypropylene glycol,polyethylene oxide, polypropylene oxide, poloxamer, polyacrylic acid,poly(lactide-co-glycolide), alginate, gelatin, collagen, fibrinogen,hydroxypropylmethylcellulose and hydroxypropyl-guar, as well asderivatives thereof, co-polymers thereof, and combinations thereof.

In a preferred embodiment the expanded cell population according to theinvention may be delivered to a recipient via a localising agent whichis a biomatrix. In a more preferred embodiment the localising agent is alight curable, degradable biomatrix. Preferably this is able to beinjected into the eye. A specific example of a biomatrix is GelMA, whichis methacrylamide modified gelatin, and is also known as gelatinmethacrylate.

GelMA may be prepared according to standard protocols known in the art(Van Den Bulcke et al., Biomacromolecules, 2000, p. 31-38; Yue et al.,Biomaterials, 2015, p. 254-271). For example, gelatin from porcine skin(gel strength 300 g Bloom, Type A) is dissolved in PBS without calciumand magnesium (Dulbeccos PBS), and methacrylic anhydride may be addedwith strong agitation into the gelatin solution to reach the desiredconcentration (e.g. 8% (vol/vol). The mixture may be stirred before andafter adding further DPBS. The diluted mixture may be purified viadialysis against Milli-Q water using dialysis tubing to removemethacrylic acid. The purified samples may optionally be lyophilized andthe solid stored at −80° C., −20° C., or 4° C. until further use.

A GelMA stock solution is prepared by dissolving lyophilized GelMA in aformulation suitable for ocular use comprising pharmaceuticallyacceptable excipients. To prepare a GelMA stock solution, lyophilizedGelMA may be dissolved in DPBS. After the GelMA is fully dissolved, aphotoinitiator (for example such as lithiumphenyl-2,4,6-trimethylbenzoylphosphinate) may be introduced into theGelMA solution. To adjust the pH to neutral, NaOH may be added to thesolution before filtering using 0.22 micrometre sterile membranes. Thefinal filtrate may be separated into aliquots and stored at 4° C. untilfurther use.

In one aspect according to the invention, the cells are encapsulatedwithin the biomatrix using a photoinitiator to polymerise the biomatrix,which is preferably GelMa. Suitable photoinitiator agents are Irgacure2959, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, sodiumphenyl-2,4,6-trimethylbenzoylphosphinate, lithiumbis(2,4,6-trimethylbenzoyl)phosphinate, sodiumbis(2,4,6-trimethylbenzoyl)phosphinate,Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, eosin Y, riboflavinphosphate, camphorquinone, Quantacure BPQ, Irgacure 819, Irgacure 1850,and Darocure 1173. In a preferred embodiment, the photoiniator islithium phenyl-2,4,6-trimethylbenzoylphosphinate, sodiumphenyl-2,4,6-trimethylbenzoylphosphinate, riboflavin phosphate. Inanother embodiment the photoinitiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate.

Prior to polymerization, the light curable biomatrix is combined with asuitable photoinitiator in a formulation suitable for ocular usecomprising pharmaceutically acceptable excipients in suitable containersknown in the art such as vials. The photoinitiator may be combined withthe biomatrix prior to mixing with cells; alternatively thephotoinitiator may be combined with the biomatrix after mixing withcells; alternatively the photoiniator may be added to the cells first,then combined with the biomatrix. The concentration of biomatrix andphotoinitiator is dependent on the specific biomatrix and specificphotoinitiator used, but is chosen to provide polymerization within aconvenient light exposure duration, typically less than about 5 minutes;preferably less than about 2 minutes; more preferably less than aboutone minute. In one embodiment the photoinitiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate and its concentration in theformulation for cell delivery to the eye is about 0.01% w/v to about0.15% w/v. In another aspect the lithiumphenyl-2,4,6-trimethylbenzoylphosphinate concentration in theformulation for cell delivery to the eye is about 0.05% w/v or about0.075% w/v. LAP may be synthesized using published procedure(Biomaterials 2009, 30, 6702-6707) and is also available from TCI (Prod.#L0290) and Biobots (BioKey).

The cells may be added to the GelMA in suitable containers known in theart such as vials or tubes. The cells may for example be added bypipetting into the GelMA and mixing by gentle pipetting up and down. Inone embodiment the GelMA concentration in the composition suitable forocular delivery is about 10 to about 200 mg/mL, or about 25 to about 150mg/mL, or about 25 to about 75 mg/mL. In a preferred embodiment theGelMA concentration in the composition suitable for ocular delivery isabout 25 mg/mL, about 50 mg/mL or about 75 mg/mL.

To polymerise the light curable biomatrix, the biomatrix,photoinitiator, and cells are exposed to a light source for a preferredduration, as described above. The wavelength of light used forpolymerization will depend on the photochemical properties of thespecific photoinitiator used. For example, photoinitation ofpolymerization for Irgacure 2959 will occur with light of wavelengthbetween 300-370 nm; photoinitation of polymerization for lithiumphenyl-2,4,6-trimethylbenzoylphosphinate will occur with light ofwavelength between 300-420 nm; photoinitation of polymerization forriboflavin-5′-phosphate will occur with light of wavelength between300-500 nm. The light source used may emit a range of wavelengths, likethat achieved with incandescent lamps, gas discharge lamps, or metalvapor lamps; alternatively, the light source used may emit a narrowrange of wavelengths, like that achieved with optical filters or with anlight emitting diode (LED). Preferably, the light source used does notemit light with wavelength less than 315 nm to avoid the damagingeffects of UV irradiation on cells. In one embodiment, the light sourceis a white light source with a spectral range of 415-700 nm. In anotherembodiment the light source is a LED light source with spectral range ofabout 365±5 nm, about 375±5 nm, about 385±5 nm, about 395±5 nm, about405±5 nm, about 415±5 nm, about 425±5 nm, about 435±5 nm, about 445±5nm, about 455±5 nm, or about 465±5 nm. The intensity of light is chosento minimize phototoxicity and provide polymerization within a convenientlight exposure duration, typically less than about 5 minutes; preferablyless than about 2 minutes; more preferably less than about one minute.One indication of polymerization is an increase in solution viscosity.Another indication of polymerization is the onset of gelation.

The polymerization of the biomatrix may occur on the ocular surface viabioprinting techniques, or alternatively on a carrier that is thentransplanted to the ocular surface. Optionally the polymerization of thebiomatrix may occur on the cornea surface in the anterior chamber, oralternatively on a carrier that is then transplanted to the corneasurface in the anterior chamber.

Carrier

The cells and localising agent suitable for ocular delivery arepreferably delivered via a carrier such as a contact lens or amnioticmembrane.

Contact lenses suitable for use according to the invention arepreferably those which conform to the patient's corneal curvature andare able to be well tolerated by the patient in clinical practice forcontinuous use as bandage contact lenses for several days.

Examples of suitable types of contact lens according to the inventionare consistent with what has been extensively validated in clinical usefor long-term bandage contact lens use with Boston keratoprosthesis type1 (which can be also used in patients with limbal stem cell deficiency)and described in: Thomas, Merina M. D.; Shorter, Ellen O. D.; Joslin,Charlotte E. O. D., Ph.D.; McMahon, Timothy J. O. D.; Cortina, M.Soledad M. D. Contact Lens Use in Patients With Boston KeratoprosthesisType 1: Fitting, Management, and Complications. Eye Contact Lens. 2015November; 41(6):334-40.

A contact lens can be of any appropriate material known in the art orlater developed, and can be a soft lens, a hard lens, or a hybrid lens,preferably a soft lens, more preferably a conventional hydrogel contactlens or a silicone hydrogel (SiHy) contact lens.

A “conventional hydrogel contact lens” refers to a contact lenscomprising a hydrogel bulk (core) material which is a water-insoluble,crosslinked polymeric material, is theoretically free of silicone, andcan contain at least 10% by weight of water within its polymer matrixwhen fully hydrated. A conventional hydrogel contact lens typically isobtained by copolymerization of a conventional hydrogel lens formulation(i.e., polymerizable composition) comprising silicone-free, hydrophilicpolymerizable components known to a person skilled in the art.

Examples of conventional hydrogel lens formulation for making commercialhydrogel contact lenses include, without limitation, alfafilcon A,acofilcon A, deltafilcon A, etafilcon A, focofilcon A, helfilcon A,helfilcon B, hilafilcon B, hioxifilcon A, hioxifilcon B, hioxifilcon D,methafilcon A, methafilcon B, nelfilcon A, nesofilcon A, ocufilcon A,ocufilcon B, ocufilcon C, ocufilcon D, omafilcon A, phemfilcon A,polymacon, samfilcon A, telfilcon A, tetrafilcon A, and vifilcon A.

A “SiHy contact lens” refers to a contact lens comprising a siliconehydrogel bulk (core) material which is a water-insoluble, crosslinkedpolymeric material containing silicone and can contains at least 10% byweight of water within its polymer matrix when fully hydrated. Asilicone hydrogel contact lens typically is obtained by copolymerizationof a silicone hydrogel lens formulation comprising at leastsilicone-containing polymerizable component and hydrophilicpolymerizable components known to a person skilled in the art.

Examples of SiHy lens formulation for making commercial SiHy contactlenses include, without limitation, asmofilcon A, balafilcon A,comfilcon A, delefilcon A, efrofilcon A, enfilcon A, fanfilcon A,galyfilcon A, lotrafilcon A, lotrafilcon B, narafilcon A, narafilcon B,senofilcon A, senofilcon B, senofilcon C, smafilcon A, somofilcon A, andstenfilcon A.

In a preferred embodiment the carrier is a contact lens selected fromthe group consisting of Balafilcon A, Lotrafilcon A, Lotrafilcon B,Senofilcon A and methafilcon A.

In a particularly preferred embodiment the carrier is a contact lens,which is Lotrafilcon B.

The carrier may be held in place on the ocular surface using fibrin glueor sutures to prevent eye movements from dislodging the construct.

The carrier combined with biomatrix and cells may be left on the eye fora range of times in order to deliver the cells, for example a few daysto one week, preferably one week.

Other Delivery Methods:

In an alternative embodiment the LSCs may be delivered as a cellsuspension to the ocular surface (without a localising agent such as abiomatrix and with/or without a carrier such as a contact lens).Compounds and excipients known in the art to improve tissue adhesionsuch as mucoadhesive agents, viscosity enhancers, or reverse thermalgelators may be included in the formulation.

Bioprinting Step

The population of ocular cells, e.g. corneal endothelial cells,obtainable according to the method of cell population expansionaccording to the invention may be grafted to the eye of a subject, e.g.,to the cornea of a subject.

The cell population according to the invention may be delivered via alocalising agent suitable for ocular use which is a light curable,degradable biomatrix such as GelMA. The following methods describeprocedures for controlling the delivery to the inner wall of the cornea.

Method 1. Bubble Depression Method (Shown in FIG. 22 )

The dysfunctional endothelial cells may first be detached from the innerwall of the cornea by peeling/scraping or in a controlled manner usingphotodisruption with a femtosecond laser. A small bolus of thecell-laden biomatrix is then injected near the interior surface of thecornea. This may be done manually using a standard syringe or customapplicator. It can also be controlled through a surgical system (e.g.constellation) or syringe pumps. A gas bubble is then injected beneaththe bolus. The gas bubble squeezes the bolus against the posteriorcornea, creating a thin coating. The entire gel is then cured using ausing a UV or near UV light source, or any other spectral band needed tocure the biomatrix. Alternatively, the dysfunctional tissue may be left,and the biomatrix cured over top of it. The light source can be focusedinto different sizes using other optical focusing methods to control thecuring area. The remaining uncured area can be flushed out usingirrigating/aspirating canula.

Method 2. Subtractive method using femtosecond laser (shown in FIG. 23 )The dysfunctional endothelial cells may first be detached from the innerwall of the cornea by peeling/scraping or in a controlled manner usingphotodisruption with a femtosecond laser. Alternatively, they may beleft in place. The cell-laden biomatrix is then injected onto theinterior surface of the cornea covering the void where tissue wasremoved or over the dysfunctional tissue. This may be done manuallyusing a standard syringe or custom applicator. It can also be controlledthrough a surgical system (e.g. constellation) or syringe pumps. Thebiomatrix is then cured using a using a UV or near UV light source, orany other spectral band needed to cure the biomatrix. The femtosecondlaser is then used to detach excess material, controlling the thicknessand area to a desired distribution. The excess material is then removedwith forceps through a corneal incision.

Method 3. Stain mask and absorption based thickness control (shown inFIG. 24 ) A biocompatible stain (Trypan Blue, Brilliant Blue, etc.) isfirstly used to dye the inner surface of the cornea. The dysfunctionalendothelial cells are then detached from the inner wall of the cornea bypeeling/scraping. The cell-laden biomatrix containing the biocompatiblestain is then injected onto the interior surface of the cornea coveringthe void where tissue was removed. The biomatrix is then cured using ausing a UV or near UV light source, or any other spectral band needed tocure the biomatrix. The stain in the corneal tissue increases the lightabsorption acting as a mask to control the area of the cured biomatrix.Similarly, the stain in the biomatrix increases the absorption of lightthereby controlling the depth/thickness of the cured material. Uncuredgel material is then flushed from the anterior chamber using anirrigating/aspirating cannula.

Method 4. Dry anterior chamber application (shown in FIG. 25 )

The dysfunctional endothelial cells may first be detached from the innerwall of the cornea by peeling/scraping or in a controlled manner usingphotodisruption with a femtosecond laser. Alternatively it may be leftin place. The anterior chamber of the anterior segment is then drainedof aqueous and replaced with gas (e.g. air). The cell-laden biomatrix isthen applied to interior surface of the cornea in small controlleddroplets (allowing surface tension to disperse the drops), or paintedusing a brush or soft tip cannula. Hyaluronic acid may be applied to thebiomatrix to alter its viscous properties and enable better control overdispensing/application. The entire biomatrix is then cured using a usinga UV or near UV light source, or any other spectral band needed to curethe biomatrix. Finally, the anterior chamber is then filled again withbalanced salt solution.

Method 5. Naturally Buoyant Formulation

The dysfunctional endothelial cells may first be detached from the innerwall of the cornea by peeling/scraping or in a controlled manner usingphotodisruption with a femtosecond laser. A small bolus of thecell-laden biomatrix is then injected near the interior surface of thecornea. The biomatrix is formulated to be naturally buoyant relative toaqueous humor or aerated to achieve the same effect. This causes thebiomatrix to naturally rise to posterior cornea, creating a thincoating. The entire biomatrix is then cured using a using a UV or nearUV light source, or any other spectral band needed to cure thebiomatrix. Alternatively, the dysfunctional tissue may be left, and thebiomatrix cured over top of it. The UV light source can be focused intodifferent sizes using optical focusing methods to control the curingarea. The remaining uncured area can be flushed out using aspirationcanula.

Other Delivery Methods

In an alternative embodiment an expanded cell population, such as CECsas described herein, may be delivered as a cell suspension (without alocalising agent such as a light curable, degradable biomatrix) and leftto attach by gravity by having the patient look down for 3 hours.Compounds and excipients known in the art to improve tissue adhesionsuch as adhesive agents, viscosity enhancers, or reverse thermalgelators may be included in the formulation.

In yet another alternative embodiment an expanded cell population, suchas CECs as described herein can also be delivered by using magneticbeads. A suspension of CECs/beads in a medium suitable for oculardelivery is prepared and this is then injected into the eye. Cellattachment is promoted by a magnet applied to the eye. (Magneticfield-guided cell delivery with nanoparticle-loaded human cornealendothelial cells. Moysidis S N, Alvarez-Delfin K, Peschansky V J,Salero E, Weisman A D, Bartakova A, Raffa G A, Merkhofer R M Jr, Kador KE, Kunzevitzky N J, Goldberg J L. Nanomedicine. 2015 April;11(3):499-509. doi: 10.1016/j.nano.2014.12.002.)

Other Methods of Use of the LATS Inhibitors According to the Invention

In one aspect according to the invention it is possible to directly addthe LATS inhibitors according to the invention directly to the surfaceof the eye to achieve cell population expansion. For example this can bedone if the cell loss was not total and there is a remaining seedingpopulation of cells to be expanded. For example if there are remaininglimbal stem cells in the eye of the patient or if there are remainingcorneal endothelial cells in the cornea of the patient as the case maybe. For example, this can be achieved by preparing the compounds asdisclosed herein in a formulation suitable for ocular use comprisingpharmaceutically acceptable excipients and applying by eye drops to theeye. Alternatively, compounds as disclosed herein in a formulationsuitable for ocular use comprising pharmaceutically acceptableexcipients can be applied intraocularly, for example, intracamerally.

Pharmaceutically acceptable excipients enhance or stabilize thecomposition, or facilitate preparation of the composition.Pharmaceutically acceptable excipients include solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like that are physiologicallycompatible.

The LATS inhibitor according to the invention may be combined withophthalmologically acceptable preservatives, co-solvents, surfactants,viscosity enhancers, penetration enhancers, buffers, sodium chloride, orwater to form an aqueous ophthalmic suspension or solution. Topicalophthalmic products may be packaged, for example, in multidose form.Preservatives may thus be required to prevent microbial contaminationduring use. Suitable preservatives include: chlorobutanol, methylparaben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbicacid, polyquaternium-1, or other agents known to those skilled in theart. Such preservatives are typically employed at a level of from 0.001to 1.0% w/v.

In an embodiment of the invention the LATS inhibitors according to theinvention could be used to preserve cells, e.g. corneal epithelial andlimbal cells until transplantation. After postmortem dissection byeye-bank specialists, corneas are preserved in media such as Optisol orPBS until transplantation. In a specific embodiment of the invention aLATS inhibitor may be added to a solution generally used for cornealtransplantation, such as Optisol or PBS. Preferably the concentration ofthe LATS inhibitor in the preservation solution is 0.5 to 100micromolar, more preferably 0.5 to 25 micromolar, particularlypreferably 1 to 20 micromolar. In a specific embodiment the LATSinhibitors according to Formula I are added at a concentration of about3 to10 micromolar.

Immunosuppressant, Anti-Inflammatory and Antibiotic Agents

In addition to, or as an alternative to, the use of a gene editingtechnique performed in vitro on a cell population, as described forlimbal stem cells or corneal endotheal cells, to mitigate the risk ofimmune rejection of the transplanted cells to the patient, the cellpopulation according to the invention may be administered simultaneouslyor sequentially with an immunosuppressant and/or anti-inflammatoryagents or agents. Standard agents for immunosuppression oranti-inflammatory agents or agents may be used including, but notlimited to, dexamethasone or cyclosporine.

Antibiotics may also be administered to the patient in conjunction withan expanded cell population (e.g. the limbal stem cells or cornealendotheal cells as described herein), such as the antibiotics cefazolinand tobramycin.

When the cell population of the present invention is administeredtogether with another agent or agents, the cell population and theagent(s) can be administered sequentially in any order orsimultaneously. In some embodiments, an expanded cell populationaccording to the invention is administered in conjunction with surgicaltreatments. In other embodiments, the limbal stem cell populationaccording to the invention is administered in conjunction with surgicaltreatments. In other embodiments, the corneal endothelial cellpopulation according to the invention is administered in conjunctionwith surgical treatments.

Therapeutic Uses

The ocular cell population according to the invention may be used in amethod of treatment or prophylaxis of an ocular disease or disordercomprising administering to a subject in need thereof of atherapeutically effective amount of a cell population comprising ocularcells.

The limbal stem cell population according to the invention may be usedin a method of treatment or prophylaxis of an ocular disease or disordercomprising administering to a subject in need thereof of atherapeutically effective amount of a cell population comprising limbalstem cells. Preferably the ocular disease or disorder is associated withlimbal stem cell deficiency.

Limbal stem cell deficiency may arise as a result of several diverseconditions including but not limited to:

-   -   direct stem cell damage from chemical or thermal burns or        radiation injury;    -   congenital conditions such as aniridia, sclerocornea, multiple        endocrine neoplasia;    -   autoimmune disorders such as Stevens Johnson syndrome or ocular        cicatricial pemphigoid or collagen vascular diseases;    -   chronic non-auto-immune inflammatory disorders such as contact        lens use, dry eye disease, rosacea, staph marginal, keratitis        (bacterial, fungal & viral), pterygia or neoplasm;    -   iatrogenic, such as after multiple eye surgeries, excision of        pterygia or neoplasm, cryotherapy;    -   as a result of medication toxicity such as preservatives        (thimerosal, benzalkonium), topical anesthetics, pilocarpine,        beta blockers, mitomycin, 5-fluorouracil, silver nitrate, and        oral medications causing Stevens Johnson syndrome.

(See: Dry Eye: a practical guide to ocular surface disorders and stemcell surgery. SLACK 2006—Rzany B, Mockenhaupt M, Baur S et al. J. Clin.Epidemiol. 49, 769-773 (1996)).

The most commonly encountered causes of limbal stem cell deficiency inclinical practice are chemical burns, aniridia, Stevens Johnson Syndromeand contact lens use.

More preferably the ocular disease or disorder is limbal stem celldeficiency which arises due an injury or disease or disorder selectedfrom the group consisting of chemical burns, thermal burns, radiationinjury, aniridia, sclerocornea, multiple endocrine neoplasia, StevensJohnson syndrome, ocular cicatricial pemphigoid, collagen vasculardiseases, chronic non-auto-immune inflammatory disorders arising fromcontact lens use, dry eye disease, rosacea, staph marginal, keratitis(including bacterial, fungal & viral keratitis), pterygia or neoplasm,limbal stem cell deficiency arising after multiple eye surgeries orexcision of pterygia or neoplasm or cryotherapy; and limbal stem celldeficiency arising as a result of medication toxicity from a medicationselected from the group consisting of preservatives (thimerosal,benzalkonium), topical anaesthetics, pilocarpine, beta blockers,mitomycin, 5-fluorouracil, silver nitrate, and oral medications causingStevens Johnson syndrome.

In a specific embodiment, the present invention provides a method oftreating limbal stem cell deficiency by administering to a subject inneed thereof an effective amount of a limbal stem cell populationobtainable by the method of cell population expansion according to theinvention.

In a more specific embodiment, the present invention provides a methodof treating limbal stem cell deficiency which arises due an injury ordisorder selected from the group consisting of chemical burns, thermalburns, radiation injury, aniridia, sclerocornea, multiple endocrineneoplasia, Stevens Johnson syndrome, ocular cicatricial pemphigoid,collagen vascular diseases, chronic non-auto-immune inflammatorydisorders arising from contact lens use, dry eye disease, rosacea, staphmarginal, keratitis (including bacterial, fungal & viral keratitis),pterygia or neoplasm, limbal stem cell deficiency arising after multipleeye surgeries, or excision of pterygia or neoplasm or cryotherapy; andlimbal stem cell deficiency arising as a result of medication toxicityfrom a medication selected from the group consisting of preservatives(thimerosal, benzalkonium), topical anesthetics, pilocarpine, betablockers, mitomycin, 5-fluorouracil, silver nitrate, and oralmedications causing Stevens Johnson syndrome by administering to asubject in need thereof a therapeutically effective amount of a limbalstem cell population obtainable by the method of cell populationexpansion according to the invention.

In yet a more specific embodiment, the present invention provides amethod of treating limbal stem cell deficiency which arises due aninjury or disease or disorder selected from the group consisting ofchemical burns, aniridia, Stevens Johnson Syndrome and contact lens useby administering to a subject in need thereof a thereapeuticallyeffective amount of a limbal stem cell population obtainable by themethod of cell population expansion according to the invention.

When an adult is a recipient (transplant recipient), preferably greaterthan 1000 p63alpha expressing cells may be administered to a patient inthe methods of treatment according to the invention. In a preferredembodiment, 1000 to 100 000 p63alpha expressing cells may beadministered to a patient in the methods of treatment according to theinvention.

The corneal endothelial cell population according to the invention maybe used in a method of treatment or prophylaxis of an ocular disease ordisorder comprising administering to a subject in need thereof of atherapeutically effective amount of a cell population comprising cornealendothelial cells. Preferably the ocular disease or disorder isassociated with decreased corneal endothelial cell density. In apreferred embodiment the ocular disease or disorder is cornealendothelial dysfunction.

More preferably the ocular disease or disorder is corneal endothelialdysfunction which is selected from the group consisting of Fuchsendothelial corneal dystrophy, bullous keratopathy (includingpseudophakic bullous keratopathy and aphakic bullous keratopathy),corneal transplant failure, posterior polymorphous corneal dystrophy,congenital hereditary endothelial dystrophy, X-linked endothelialcorneal dystrophy, aniridia, and corneal endothelitis. In a specificembodiment the ocular disease or disorder is selected from the groupconsisting of Fuchs endothelial corneal dystrophy, bullous keratopathy(including pseudophakic bullous keratopathy and aphakic bullouskeratopathy) and corneal transplant failure.

In a specific embodiment, the present invention provides a method oftreating corneal endothelial dysfunction by administering to a subjectin need thereof an effective amount of a corneal endothelial cellpopulation obtainable by the method of cell population expansionaccording to the invention.

In a more specific embodiment, the present invention provides a methodof treating corneal endothelial dysfunction which is selected from thegroup consisting of Fuchs endothelial corneal dystrophy, bullouskeratopathy (including pseudophakic bullous keratopathy and aphakicbullous keratopathy), corneal transplant failure, posterior polymorphouscorneal dystrophy, congenital hereditary endothelial dystrophy, X-linkedendothelial corneal dystrophy, aniridia, and corneal endothelitis byadministering to a subject in need thereof an effective amount of acorneal endothelial cell population obtainable by the method of cellpopulation expansion according to the invention.

In yet a more specific embodiment, the present invention provides amethod of treating corneal endothelial dysfunction selected from thegroup consisting of Fuchs endothelial corneal dystrophy, bullouskeratopathy (including pseudophakic bullous keratopathy and aphakicbullous keratopathy) and corneal transplant failure by administering toa subject in need thereof an effective amount of a corneal endothelialcell population obtainable by the method of cell population expansionaccording to the invention.

When an adult is a recipient (transplant recipient), the cornealendothelial cell layer for use in the method of treatment according tothe invention preferably has a final cell density in the eye of about atleast 500 cells/mm² (area), preferably 1000 to 3500 cells/mm² (area),more preferably 2000 to about 4000 cells/mm² (area).

EXAMPLE SECTIONS A-C

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the disclosure belongs.

Example A1: LATS1 Biochemical Assay: Homogenous Time ResolvedFluorescence (HTRF) Assay (Compound Examples 1-290)

The LATS1 biochemical HTRF assay was performed using the HTRFKinEASE-STK S1 kit (CisBio, catalogue number 62ST1PEC) according tomanufacturer's instructions. Human LATS1 kinase domain protein waspurchased from Carnabio (catalogue number 01-123), which harbors thecatalytic domain of amino acids 589-1130, and was co-purified with humanHis-tagged MOBKL1A (NP_775-739). Compounds were added by ECHO liquidhandler (Labcyte) into 384-well plates. Then 5 microlitre of thefollowing solution was added into the wells (50 mM HEPES, 0.01% BSA, 100nM Orthaovanadate, 1 mM MgCl2, 1 mM DTT, 0.6 ng/microlitre LATS1 enzyme(Carnabio, 01-123), 2 micromolar STK1 localising aid (CisBio)), followedwith 5 microlitre of 2 mM ATP in the kinase buffer (50 mM HEPES, 0.01%BSA, 100 nM Orthaovanadate, 1 mM MgCl2, 1 mM DTT) and incubated for 30minutes at room temperature. 10 microlitre of detection mix (50 mMHEPES, 0.01% BSA, 100 nM Orthaovanadate, 1 mM MgCl2, 1 mM DTT, STKAntibody-Cryptate (CisBio), Streptavidin-XL665 (CisBio), 500 micromolarpotassium fluoride, 50 nM EDTA) was added to each well and incubated for1 hour at room temperature. Plates were read on Pherastar (BMG Labtech)for HTRF (665 nm/620 nm). The IC₅₀ is measured when the effect of thecompound reduces the HTRF signal by 50%. LATS1 IC₅₀ values for theexemplified compounds are shown in Table 1A.

Example A2: LATS1 Biochemical Caliper Assay

Human LATS1 kinase domain protein was purchased from Carnabio (cataloguenumber 01-123), which harbors the catalytic domain of amino acids589-1130, and was co-purified with human His-tagged MOBKL1A(NP_775-739). 5 microlitre of enzyme buffer, 100 nL of compounds and 5microlitre of localising aid buffer were added into 384-well plates. Thefinal assay reaction mix contains 100 mM HEPES, pH7.5, 0.1% BSA, 0.01%Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 micromolar Sodium Orthovanadate,10 micromolar Beta-Glycerophosphate, 400 micromolar ATP, 1% DMSO, 1.1 nMLATS1 enzyme (Carnabio, 01-123), 1 micromolar localising aid ofFAM-KKLRRTLSVA-COOH (SEQ ID NO: 24) (NanoSyn). The plates were incubatedat 25° C. for 3 hours. 40 microlitre of stop buffer with 25 mM EDTA(NanoSyn) was added to each well to terminate the reaction. Localisingaids and products were separated electrophoretically using themicrofluidic-based Caliper Labchip 3000 Drug Discovery System (CaliperLife Sciences). Plates were read using blue laser excitation and greenfluorescence detection and quantified by fluorescence intensity. TheIC₅₀ is measured when the effect of the compound reduces the productfluorescence signal by 50%. According to this assay the LATS1 IC₅₀values in micromolar for the following compounds are:

Ex. 49: 0.012, Ex. 14: 0.034, Ex. 65: 0.022, Ex, 139: 0.028, Ex. 133:0.004. Example A3: LATS2 Biochemical Caliper Assay (Compound Examples1-290)

Human LATS2 kinase domain protein was purchased from Carnabio (cataloguenumber 01-124), which harbors the catalytic domain of amino acids553-1088, and was co-purified with human His-tagged MOBKL1A(NP_775-739). 5 microlitre of enzyme buffer, 100 nL of compounds and 5microlitre of localising aid buffer were added into 384-well plates. Thefinal assay reaction mix contains 100 mM HEPES, pH7.5, 0.1% BSA, 0.01%Triton X-100, 1 mM OTT, 10 mM MgCl2, 10 micromolar Sodium Orthovanadate,10 micromolar Beta-Glycerophosphate, 400 micromolar ATP, 1% DMSO, 1.1 nMLATS2 enzyme (Carnabio, 01-124), 1 micromolar localising aid ofFAM-KKLRRTLSVA-COOH (SEQ ID NO: 24) (NanoSyn). The plates were incubatedat 25° C. for 3 hours. 40 microlitre of stop buffer with 25 mM EDTA(NanoSyn) was added to each well to terminate the reaction. Localisingaids and products were separated electrophoretically using themicrofluidic-based Caliper Labchip 3000 Drug Discovery System (CaliperLife Sciences). Plates were read using blue laser excitation and greenfluorescence detection and quantified by fluorescence intensity. TheIC₅₀ is measured when the effect of the compound reduces the productfluorescence signal by 50%. LATS2 IC₅₀ values for the exemplifiedcompounds 1-290 are shown in Table 1A.

TABLE 1A Inhibitory Activity against LATS1 and LATS2 Example LATS1 LATS2No. IC₅₀ (μM) IC₅₀ (μM)  1 0.001 n.d.  2 0.001 n.d.  3 0.001 n.d.  40.001 0.001  5 0.002 n.d.  6 0.001 0.008  7 0.002 n.d.  8 0.003 n.d.  90.003 n.d.  10 0.003 n.d.  11 0.003 0.012  12 0.004 0.015  13 0.004 n.d. 14 0.002 0.05  15 0.004 n.d.  16 0.005 n.d.  17 0.005 n.d.  17a 0.003n.d.  17b 0.0009 n.d.  17c 0.003 n.d.  17d 0.001 n.d.  18 0.006 n.d.  190.006 n.d.  20 0.006 n.d.  21 0.007 n.d.  22 0.007 n.d.  23 0.01 n.d. 24 0.017 n.d.  25 0.03 n.d.  26 0.031 n.d.  27 0.035 n.d.  28 0.036n.d.  29 0.036 n.d.  30 0.039 n.d.  31 0.041 n.d.  32 0.047 n.d.  330.071 n.d.  34 0.095 n.d.  35 0.118 n.d.  36 0.199 n.d.  37 0.233 n.d. 38 0.24 n.d.  39 0.244 n.d.  40 0.328 n.d.  41 0.708 n.d.  42 0.711n.d.  43 >1.97 n.d.  44 >2.5 n.d.  45 >2.5 n.d.  46 >2.5 n.d.  47 0.0020.006  48a 0.002 0.002  48b 0.006 0.018  49 0.001 0.021  50 0.005 n.d. 51 0.006 n.d.  52 0.012 n.d.  53 0.04 n.d.  54 0.078 n.d.  55 0.46 n.d. 56 1.16 n.d.  57 1.44 n.d.  58 0.001 0.17  59 0.001 0.004  60 0.001n.d.  61 0.001 n.d.  62 0.001 0.002  63 0.001 n.d.  64 0.001 n.d.  650.002 0.03  66 0.002 0.011  67 0.002 n.d.  68 0.002 n.d.  68a 0.001 n.d. 68b 0.002 n.d.  69 0.003 n.d.  70 0.003 n.d.  71 0.004 n.d.  72 0.004n.d.  73 0.006 n.d.  74 0.006 n.d.  75 0.008 n.d.  76 0.009 n.d.  770.009 n.d.  78 0.011 n.d.  79 0.011 n.d.  80 0.011 n.d.  81 0.011 n.d. 82 0.013 n.d.  83 0.017 n.d.  84 0.021 n.d.  85 0.025 n.d.  86 0.026n.d.  87 0.03 n.d.  88 0.037 n.d.  89 0.044 n.d.  90 0.126 n.d.  910.148 n.d.  92 0.304 n.d.  93 0.809 n.d.  94 1.15 n.d.  95 >1.44 n.d. 96 >1.70 n.d.  97 >2.5 n.d.  98 1.35 n.d.  99 0.001 n.d. 100 0.004 n.d.101 0.005 0.03 102 0.126 n.d. 103 0.002 n.d. 104 0.005 n.d. 105 0.001n.d. 106 0.003 n.d. 107 0.008 n.d. 108 0.027 n.d. 109 0.001 n.d. 1100.002 n.d. 111 0.001 n.d. 112 0.002 n.d. 113 0.235 n.d. 114 0.002 n.d.115 0.002 n.d. 116 0.003 n.d. 117 0.004 n.d. 118 0.005 n.d. 119 0.009n.d. 120 0.01 n.d. 121 0.014 n.d. 122 0.015 n.d. 123 0.018 n.d. 1240.022 n.d. 125 0.023 n.d. 126 0.053 n.d. 127 0.055 n.d. 128 0.077 n.d.129 0.187 n.d. 130 1.153 n.d. 131 >0.833 n.d. 132 >2.5 n.d. 133 0.0020.004 134 0.001 n.d. 135 0.002 n.d. 136 0.002 n.d. 137 0.022 n.d. 1380.002 n.d. 139 0.003 0.02 140 0.004 n.d. 141 0.004 n.d. 142 0.006 n.d.143 0.007 n.d. 144 0.01 n.d. 145 0.012 n.d. 146 0.033 n.d. 147 0.069n.d. 148 0.168 n.d. 149 0.171 n.d. 150 4.27 n.d. 151 >10 n.d. 152 >2.5n.d. 153 >5 n.d. 154 0.0 n.d. 155 0.001 n.d. 156 0.006 n.d. 157 0.006n.d. 158 0.008 n.d. 159 0.016 n.d. 160 0.024 n.d. 161 0.025 n.d. 1620.049 n.d. 163 0.051 n.d. 164 0.159 n.d. 165 >2.5 n.d. 166 0.014 n.d.167 0.143 n.d. 168 0.028 n.d. 169 0.098 n.d. 170 1.28 n.d. 171 0.054n.d. 172 0.017 n.d. 173 0.061 n.d. 174 0.197 n.d. 175 0.324 n.d.176 >2.5 n.d. 177 >2.5 n.d. 178 >2.5 n.d. 179 0.002 n.d. 180 0.322 n.d.181 9.0 n.d. 182 >10 n.d. 183 0.797 n.d. 184 0.004 n.d. 185 0.009 n.d.186 0.009 0.05 187 0.016 n.d. 188 0.017 n.d. 189 0.083 n.d. 190 0.099n.d. 191 0.189 n.d. 192 0.201 n.d. 193 >10 n.d. 194 0.005 n.d. 195 >10n.d. 196 >2.5 n.d. 197 0.023 n.d. 198 0.001 n.d. 199 0.001 n.d. 2510.004 n.d. 252 0.002 n.d. 253 0.085 n.d. 254 0.005 n.d. 255 0.008 n.d.256 0.008 n.d. 257 0.022 n.d. 258 0.023 n.d. 259 0.061 n.d. 260 0.459n.d. 261 0.001 0.004 262 0.003 n.d. 263 0.007 n.d. 264 0.007 n.d. 2650.008 n.d. 266 0.009 n.d. 267 0.013 n.d. 268 0.018 n.d. 269 0.001 n.d.270 0.002 n.d. 271 0.002 n.d. 272 0.002 n.d. 273 0.463 n.d. 274 0.013n.d. 275 0.011 n.d. 276 0.017 n.d. 277 0.065 n.d. 278 0.09 n.d. 279 >2.5n.d. 280 0.003 n.d. 281 0.004 n.d. 282 0.01 n.d. 283 0.02 n.d. 284 0.03n.d. 285 0.19 n.d. 286 0.56 n.d. 287 0.002 0.015 288 0.004 0.034 2890.004 n.d. 290 0.004 0.008 n.d. means not determined

Example A4: LATS1 Biochemical Caliper Assay (Compound Examples 291-335)

The LATS1 biochemical Caliper assay was performed as following.

Human LATS1 kinase domain protein was purchased from Carnabio (cataloguenumber 01-123; lot 15CBS-0098D). Human LATS1, catalytic domain[589-1130(end) amino acids of accession number NP_004681.1] wasco-expressed as N-terminal GST-fusion protein (90 kDa) with humanHis-tagged MOBKL1A [1-216(end) amino acids of accession numberNP_775739.1] using baculovirus expression system. GST-LATS1 was purifiedby using glutathione sepharose chromatography. The substrate(Fluo-SGKtide; Peptide for LATS1; lot BS-41067) has the followingsequence: 5-Fluo-Nva-KKRNRRLSVA-amide (SEQ ID NO: 27)×TFA and waspurchased from Biosyntan.

The reaction is performed in reaction buffer containing 50 mM Hepes pH7, 5; 0.02% Tween20; 0.02% BSA; 1 mM DTT; 10 uM Na₃VO₄ and 10 mMbeta-Glycerolphosphat and fresh added 1 mM MgCl₂ and qsp H₂O.

The substrate solution (2×conc.) in Reaction Buffer contains 300 μM ATPand 4 μM Fluo-SGKtide.

The kinase solution (2×conc.) in Reaction Buffer contains 20 nM LATS1kinase.

4.5 μL of 2×conc. Kinase solution, 50 nL of 1.8 mM compounds and 4.5 μLof substrate solution were added into 384-well plates black small volumefrom Greiner and incubated at 32° C. for 1 hour. 15 μL of stop buffercontaining 100 mM Hepes pH 7, 5; 5% DMSO; 0.1% Coating reagent; 10 mMEDTA and 0.02% Brij35 and qsp H₂O to each well to terminate thereaction.

Substrates and products were electrophoretically separated using themicrofluidic-based Caliper EZ Reader System (Caliper Life Sciences)using a 12 sipper chip (cat 760404). The separation takes place inCoating Buffer (idem Stop buffer) and containing 0.1% Coating reagentCR3 and 0.5% coating reagent CR8 (Perkin Elmer).

Plates were read using a LED with an excitation at 488 nm and adetection at 520 nm to quantify the fluorescence intensity. The IC₅₀ ismeasured when the effect of the compound reduces the productfluorescence signal by 50%. LATS1 IC₅₀ values for the exemplifiedcompounds 291-335 are shown in Table 1B.

TABLE 1B Inhibitory Activity against LATS1 LATS1 Example No. IC₅₀ (μM)291 0.4570 292 0.0433 293 0.0160 294 0.0045 295 0.0100 296 0.2089 2970.0075 298 0.0590 299 0.0971 300 0.0012 301 0.0052 302 0.0595 303 0.0102304 0.0096 305 0.6629 306 0.0012 307 0.0019 308 0.0687 309 0.0008 3100.1639 311 1.0336 312 0.0024 313 0.0038 314 0.4667 315 0.0050 316 0.0425317 0.0008 318 0.0018 319 0.0581 320 0.0007 321 0.0038 322 0.0292 3230.0045 324 0.0104 325 0.0037 326 0.0572 327 0.1251 328 0.0189 329 0.0030330 0.0031 331 0.0105 332 0.0076 333 0.0023 334 0.0061 335 n.t. n.t.:not tested

Example A5: Mouse Hepatic Progenitor Cell Proliferation Assay

The proliferation assay consists in measuring the compound inducedthree-dimensional growth of hepatic progenitor cells (HPC) into smallorganoids by means of high content imaging methods. The progenitor cellswere isolated from C571BI/6 wild type mice and expanded as described(Lu, W. Y. et al., Nat. Cell Biol. 17, 971-983 (2015)). Cells arestocked in liquid nitrogen, thawed on need and tested using thefollowing protocol:

HPC are harvested from their collagen I coated culture vessel (Corning;cat. Number 354487), counted and assessed for viability using a CEDEXcell counter (Roche). After centrifugation, 12 million cells arere-suspended in 2 ml of William's E Medium (Life Technologies; cat.Number 22551089) supplemented with 10% Fetal Bovine Serum (Amimed; cat.Number 2-01F10-I); 1% Penicillin/Streptomycin (Sigma; cat. Number15140-122); 17.6 mM of NaHCO₃ (Sigma; cat. Number S8761); 20 mM HEPES(Gibco; cat. Number H3375); 10 mM Nicotinamide (Sigma; cat. NumberN0636-100); 14 mM glucose (Sigma; cat. Number G7021); 1 mM SodiumPyruvate (Sigma; cat. Number TMS-005); Insulin Transferrin Selenium(ITS) diluted 100 times from stock solution (Sigma; cat. Number 13148);100 nM Dexamethasone (Sigma; cat. Number D4902); 0.2 mM ascorbic acid(Sigma; cat. Number A7506-100G); 10 ng/ml recombinant human IL-6(Preprotech; cat. Number 200-06); 10 ng/ml murine HGF (Preprotech; cat.Number 315-23; Lot. 0711S527); 10 ng/ml murine EGF (Preprotech; cat.Number 315-09; Lot. 0217179-1). Six milliliters of Matrigel (Corning;cat. Number 354277; Lot: 5187006) are added to the cell/media solution,thus the final cell concentration is 1.5 million HPC per milliliter, in25% culture medium and 75% Matrigel. Five microliter (μl) of thecell/Matrigel suspension are transferred in each well of the clearbottom 96 well assay plate (Corning; cat. Number 356649) using the Stardispenser (Hamilton). After 20 minutes incubation at 37° C. and 5% CO₂to allow for Matrigel polymerization, 45 μl of cell culture media aredispensed on top.

In the compound source plate, 300 μl of cell culture media are dispensedon 1.2 μl of compounds dissolved in 90% DMSO. After proper mixing, 50 μlof the compound solution is transferred to the assay plate using a CyBiWell (CyBio). Each compound is tested in 8-point concentration curveshaving a dilution factor of 3.16 between each concentration. The maximumcompound concentration tested is 20 μM and the lowest is 9 nM. The assayplates are incubated for 4 days at 37° C. and 5% CO₂.

Following the incubation, the organoids are fixed with phosphate buffersaline (PBS) (Gibco; cat. Number 10010-015) containing 4%Paraformaldehyde (Electron Microscopy Sciences; cat. Number 15714-S) andHoechst 33342 (Life Technologies; cat. Number H3570) diluted 1/5000 ofthe stock solution to stain the nuclei. The plates are incubated 90minutes at room temperature and washed once with 300 μl PBS containing1% Penicillin/Streptomycin.

Plates are imaged with the CV7000 imager (Yokogawa) at a 10 foldmagnification using the bright field lamp (lamp power: 10%; exposuretime: 20 ms) and the 405 nanometer laser (laser power: 100%; exposuretime 200 ms). Four different images per well were acquired and analyzedwith the Yokogawa image analysis software (YAS). The output feature usedto determine organoid size is the mean number of nuclei per organoid,averaged on the well. The mean number of nuclei per organoid (x) isnormalized to the neutral control treatment (DMSO), using the followingcalculation: [xn=+100 (x−NC)/NC]. The neutral control-normalized dataare used for automated curve fitting to derive curve parameters percompound, including EC50 values.

The mouse hepatic progenitor cell proliferation assay may easily be usedto test whether a compound is effective at inducing hepatic progenitorcell proliferation. Certain compounds of the invention were tested inthe mouse hepatic progenitor cell proliferation assay and found to haveEC₅₀ values of less than 20 μM, e.g. Examples 48a and 58 were found tohave EC₅₀ values of 0.26 and 0.84 μM respectively. Certain othercompounds of the invention were tested in the mouse hepatic progenitorcell proliferation assay and found to have EC₅₀ values of greater than20 μM, e.g. Example 303.

Example A6: pYAP HTRF Assay (HaCaT Cells)

The pYAP HTRF assay was performed using the Phospho-YAP (SER127) 10000test kit (CisBio, catalogue number 64YAPPEH) according to manufacturer'sinstructions. Briefly, HaCaT cells were suspended at 1.1×10⁶ cells/mL inDMEM (no phenol red)+10% FBS with penicillin-streptomycin-glutamine. 5μL of cells were dispensed into 1536 well white solid bottom, tissueculture treated plates (5500 cells/well), and incubated for 48 hours at37° C. 50 nL test compound was transferred into the assay platescontaining HaCaT cells using Pintool (GNF) and incubated for 2 hours.Lysis buffer (CisBio) was added to the assay wells and incubated for 5minutes at room temperature, followed by the addition of 2 μL detectionantibodies (1:40 dilution of each pYAP d2 ab and pYAP cryptate antibodystocks). The plates were incubated overnight (20 hrs) at roomtemperature covered with metal lids. Plates were read on Pherastar (BMGLabtech) for HTRF (665 nm/620 nm). The IC₅₀ is measured when the effectof the compound reduces the HTRF signal by 50%. Results are shown inTable 1C.

Example A7: YAP Translocation Assay (HaCaT Cells)

HaCaT cells were suspended at 0.4×10⁶ cells/ml in DMEM+10% FBS withpenicillin-streptomycin-glutamine. 50 μl of cells were dispensed into384-well clear bottom assay plates (20,000 cells/well), and incubatedovernight at 37° C. 100 nL test compound was transferred into the assaywells containing HaCaT cells using ECHO (Labcyte). After 24 hoursincubation at 37° C., 7 μL of 32% PFA was dispensed into each well andincubated for 45 minutes at room temperature. Plates were washed 3 timeswith PBS, leaving 20 μL of PBS per well, then treated with 0.1% Tritonand 1.5% BSA in PBS for 45 minutes at room temperature. Plates werewashed for 5 times with PBS, leaving 20 μL of PBS per well. 20 μL of1:1000 primary YAP ab (Santa Cruz Biotechnology, catalogue number 63.7)and 1:1500 Draq5 in PBS with 1.5% BSA was added to each well andincubated for 4 hrs at room temperature. After 3 times PBS wash, leaving20 μL of PBS per well, each well was incubated with 20 μL of 1:2000AlexaFluor 488_A21202 secondary antibody (Molecular Probes) for 4 hrs atroom temperature. Plates were further washed 3 time with PBS, sealed andimaged on the high-content Opera imaging system (PerkinElmer). Resultsare shown in Table 1C.

TABLE 1C Inhibitor of phosphorylation of YAP and YAP NuclearTranslocation HaCaT nuclear HaCaT pYAP translocation Example No. IC₅₀(μM) EC₅₀ (μM)  1 0.019 0.326  2 0.067 0.584  3 0.853 0.641  4 1.15 2.53 5 2.19 2.48  6 1.24 1.70  7 0.165 0.733  8 0.198 1.20  9 2.10 2.42  102.96 3.55  11 0.646 1.43  12 1.15 1.75  13 13.45 8.51  14 7.31 4.25  150.66 3.48  16 9.79 15.8  17 0.606 2.63  17a 4.95 4.1  17b 0.765 1.22 17c 4.28 4.68  17d 0.402 1.72  18 1.52 3.46  19 24.8 17.6  20 1.77 5.18 21 19.2 8.81  22 11.4 13.0  23 9.58 11.8  24 34.7 33.1  25 n.d. >20  2654.6 >100  27 n.d. >14.4  28 n.d. >20  29 n.d. >20  30 n.d. >20  31n.d. >20  32 >10 >20  33 >20 >20  34 n.d. >20  35 n.d. >20  36 n.d. >20 37 n.d. >6.67  38 >10 >20  39 n.d. >11.6  40 >10 >20  41 >10 >20  42n.d. 11.6  43 n.d. >20  44 >10 >20  45 n.d. >20  46 n.d. >20  47 0.551.31  48a 0.37 0.80  48b 7.25 7.22  49 0.607 1.17  50 17.0 3.81  51 57.84.39  52 11.3 >20  53 n.d. >20  54 n.d. >20  55 >10 >20  56 >10 >20  57n.d. >20  58 0.185 0.302  59 0.019 0.341  60 n.d. 0.44  61 0.141 0.517 62 0.040 0.538  63 0.131 0.338  64 0.063 0.479  65 0.895 1.99  66 0.5341.01  67 n.d. 0.685  68 1.23 3.39  68a 1.43 1.68  68b 4.27 2.92  69 4.7211.5  70 0.422 1.68  71 0.026 1.57  72 1.17 3.65  73 9.07 8.26  74 3.412.7  75 3.8 6.91  76 6.42 9.41  77 >100 13.4  78 12.1 14.3  79 0.7653.87  80 21.6 12.9  81 42.8 17.0  82 >100 >20  83 42.1 28.2  84 65.2 >20 85 31.0 9.81  86 56.0 >20  87 51.6 >20  88 69.8 29.3  89 >100 >100  90n.d. >20  91 >10 >20  92 n.d. >20  93 n.d. >20  94 n.d. >20  95 >10 >20 96 n.d. >20  97 n.d. >20  98 n.d. n.d.  99 0.113 0.606 100 11.6 4.83101 5.4 >20 102 n.d. n.d. 103 2.64 2.27 104 2.6 5.19 105 19.9 >20 10614.2 >20 107 31.2 20.0 108 11.8 >19.3 109 0.977 4.71 110 2.97 15.29 1110.33 2.70 112 1.21 3.52 113 n.d. >20 114 0.565 1.33 115 1.05 1.66 1160.782 2.18 117 2.31 4.47 118 2.05 5.01 119 n.d. 5.18 120 2.14 2.93 121n.d. 8.17 122 7.4 5.8 123 9.45 9.2 124 8.01 12.0 125 n.d. 8 126 n.d. >20127 n.d. >20 128 n.d. >20 129 n.d. >20 130 n.d. >20 131 n.d. >20 132n.d. >20 133 0.414 1.67 134 0.539 1.19 135 0.488 0.85 136 1.14 2.96 13735.5 >17.8 138 3.37 9.07 139 0.38 2.66 140 1.24 2.26 141 13.56 >10142 >100 >28.02 143 >10 >20 144 0.606 4.44 145 >100 9.31 146 n.d. >20147 >10 >20 148 n.d. >20 149 >10 >20 150 n.d. n.d. 151 n.d. n.d. 152n.d. >20 153 >10 >20 154 2.53 1.06 155 1.58 1.05 156 >100 90.1 157 4.053.78 158 11.2 6.29 159 >10 >20 160 6.8 >20 161 >100 >20 162 n.d. >20 163n.d. >20 164 n.d. >20 165 n.d. >20 166 n.d. 8.45 167 n.d. >20 168 26.511.6 169 n.d. n.d. 170 >10 >20 171 n.d. 17.2 172 >10 >20 173 n.d. >20174 >10 >20 175 >10 >20 176 n.d. >20 177 n.d. >20 178 n.d. >20179 >10 >20 180 >10 >20 181 n.d. >10 182 n.d. >20 183 n.d. >20 184 2.512.81 185 n.d. 11.5 186 n.d. >20 187 >100 >20 188 >100 12.0 189 n.d. >10190 n.d. >20 191 n.d. n.d. 192 >10 >20 193 n.d. n.d. 194 11.3 4.24 195n.d. n.d. 196 >10 >20 197 n.d. n.d. 198 2.18 3.23 199 n.d. 9.1 251 1.9610.87 252 6.98 >10 253 16.67 >20 254 2.91 4.63 255 3.18 5.39 256 11.0715.6 257 n.d. >20 258 n.d. 9.92 259 n.d. 17.31 260 n.d. >20 261 0.2021.22 262 n.d. 0.311 263 3.22 5.99 264 14.8 11.2 265 22.3 >20 266 n.d.5.35 267 7.19 9.75 268 n.d. >20 269 0.294 0.438 270 0.423 0.873 2710.187 1.07 272 3.51 5.97 273 >100 >20 274 n.d. 17.3 275 n.d. 15.1 276n.d. >20 277 n.d. >20 278 n.d. >20 279 n.d. >20 280 0.476 2.73 281 0.6562.89 282 8.27 11.32 283 8.25 14.19 284 46.46 >41.19 285 n.d. >20 286n.d. >20 287 2.99 3.88 288 18.3 7.42 289 34.1 6.50 290 n.d. 1.39 n.d.means not determined

Example A8: Target Identification

siRNA against MST1/2 or LATS1/2 were transfected into human HaCaT cells.Forty-eight hours later, cells were treated with 1 μM of Okadaic acidfor two hours to enhance pYAP signals. Cell lysates were subjected towestern blot analysis for pYAP (Ser127) described below. The results ofthe experiment are reported in FIGS. 33A to 33C.

Western Blot Analysis

Cell lysates (25 μg per lane) were mixed with XT sample buffer andreducing agent (Bio-Rad), separated by 4-12% gradient SDS Criterionprecast gel (Bio-Rad), and transferred to a nitrocellulose membrane(Bio-Rad). Proteins were detected with primary antibodies andhorseradish peroxidase-conjugated secondary antibodies by using anenhanced chemiluminescence kit (ThermoFisher). Primary antibodies usedwere anti-pYAP antibody (Cell Signaling, catalogue number 13008S) andanti ACTIN antibody (Abcam, catalogue number ab8227)

Example A9: In Vivo Pharmacodynamics (PD)

8-10 week old male C57BL6 mice (Envigo) were used for the in vivo PDstudy. A sterile surgical 6 mm punch biopsy tool was used to make twofull-thickness excisional wounds on the dorsum of the anesthetizedmouse. LATS inhibitors were formulated in 49.5% propylene glycol/0.5%Tween80/49% PBS/1% HPMC, and administered topically at a dosing volumeof 3 μL. The wound area was covered with an adhesive dressing(Tegaderm). The animals were then wrapped with self-adhesive bandage.After two daily doses, the skin samples around the wound edge (2 mmring-shaped samples) were collected using scissors 7 hour after the lastdose.

The harvested samples were subjected to RNA extraction using the RNeasykit (Qiagen), according to the manufacturer's instruction. Two-StepTaqMan RT-PCR analysis was performed on a PTC-200 peltier thermal cycler(MJ Research) and an ABI PRISM 7900HT Sequence Detection system (AppliedBiosystems). cDNA was synthesized using a High-Capacity cDNA Archive kit(Applied Biosystems) according to the manufacturer's instructions.TaqMan analyses were performed using TaqMan Universal Master mix(Applied Biosystems) and Cyr61 and Gapdh probes (Applied Biosystems)according to the manufacturer's instructions. mRNA expression levels forthe target genes were normalized to Gapdh mRNA levels and data analyzedusing SDS 2.0 software (Applied Biosystems) to calculate relative RNAquantities.

All animal studies were conducted at the Genomics Institute of theNovartis Research Foundation (GNF). The experimental protocols were incompliance with animal welfare regulations and approved by theInstitutional Animal Care and Use Committee at GNF.

The resulted relative Cyr61/Gapdh expression levels against testcompound concentrations were plotted as a bar graph in FIG. 34 .

Example A10: In Vivo Histology and Ki67 Staining

7 week old male C57BL6 mice were used for the in vivo histology study.LATS inhibitors were formulated in 70% PG/30% EtOH, and administeredtopically at a dosing volume of 25 μL (250 μg per dosge) to the shaveddorsal surface. After 3 days of twice a day dosing, the skin sampleswere collected, subjected to immuno-histology staining for Ki67(ThermoFisher Scientific, Catalogue number RM-9106). The sections wereobserved visually, and Ki67 positive cells were counted by an in-houseimaging algorithm. Representative micrographs of Ki67 staining wereshown in FIG. 35A. The abundance of Ki67 positive cells in untreated andtreated mouse skin cell were plotted as a scatter plot in FIG. 35B.

Example A11: pYAP HTRF Assay (JHH-5 Cells)

The pYAP HTRF assay is performed using compound-treated JHH-5 cells(Fujise et al., Hepatogastroenterology. 1990 October; 37(5):457-60) thatare analyzed with the Phospho-YAP (Ser127) 50000 test kit (CisBiocatalog number 64YAPPEI) according to manufacturer's instructions.Briefly, JHH-5 cells are suspended at 0.48×10{circumflex over ( )}6cells/ml in William's E medium without phenol-red (Thermo FisherScientific Gibco catalog number A1217601) supplemented with 10% (v/v)heat-inactivated fetal bovine serum (Thermo Fisher Scientific Gibcocatalog number 16140071) and penicillin-streptomycin at 100 U/ml each(Thermo Fisher Scientific Gibco catalog number 15140122). Cells aredispensed into 384 well clear bottom culture plates (50 ul/well, at24000 cells per well, Greiner catalog number 781091), and incubated for24 hours in a cell culture incubator at 37° C. Test compounds aretransferred into culture plates containing JHH-5 cells using an Echo550(Labcyte) acoustic dispenser (50 nl/well), followed by incubation for 2hours at 37° C. Lysis buffer (part of CisBio kit catalog number64YAPPEI) is added to the assay plates as a four-fold concentrate(corresponding to 16 ul/well) and incubated for 30 minutes at roomtemperature with agitation. Lysate is transferred from culture platesinto white low-volume 384 well assay plates (12 ul/well, Greiner catalognumber 784075), followed by the addition of 3 ul/well of detectionantibodies (part of CisBio kit catalog number 64YAPPEI: 1:40 dilution ofeach pYAP d2 antibody and pYAP cryptate antibody stocks). The plates aresealed and incubated overnight (18 hours) at room temperature protectedfrom light. Plates are read on an EnVision plate reader (Perkin Elmer)at emission wavelengths of 665 nm and 620 nm. The HTRF signal ratio xper well is calculated as [x=Signal(665 nm)/Signal(620 nm)×10000] asspecified in the manufacturer's protocol. The signal ratio x per well isnormalized to active control and neutral control treatment (DMSO),scoring median NC as 0% and median AC as −100% to calculate normalizedsignal ratio xn per well as [xn=±100 (x−NC)/(AC−NC)].

The control-normalized data is used for automated curve fitting toderive curve parameters per compound, including IC₅₀ values. pYAP IC₅₀values in JHH-5 cells for the exemplified compounds are shown in Table1D.

Table 1D. Inhibition of phosphorylation of YAP in JHH5 cells

TABLE 1D Inhibition of phosphorylation of YAP in JHH5 cells JHH-5 pYAPExample No. IC₅₀ (μM)  1  2 >10    3 1.0  4 >10    5 2.3  6 2.6  7 1.6 8 1.9  9 n.d.  10 8.7  11 n.d.  12 2.9  13 n.d.  14 >10    15 4.1  16n.d.  17 n.d.  18 n.d.  19 n.d.  20 n.d.  21 n.d.  22 n.d.  23 n.d.  24n.d.  25 n.d.  26 n.d.  27 n.d.  28 n.d.  29 n.d.  30 >10    31 n.d.  32n.d.  33 n.d.  34 n.d.  35 n.d.  36 n.d.  37 n.d.  38 n.d.  39 n.d.  40>10    41 n.d.  42 n.d.  43 n.d.  44 n.d.  45 n.d.  46 n.d.  47 n.d. 48a 0.6  48b n.d.  49 1.6  50 >10    51 n.d.  52 n.d.  53 n.d.  54 n.d. 55 n.d.  56 n.d.  57 n.d.  58 0.6  59 1.1  60 n.d.  61 1.4  62 0.6  63n.d.  64 2.1  65 5.8  66 6.2  67 n.d.  68 3.5  69 n.d.  70 4.0  71 >10   72 4.6  73 n.d.  74 n.d.  75 n.d.  76 n.d.  77 n.d.  78 n.d.  79 2.1 80 n.d.  81 n.d.  82 n.d.  83 n.d.  84 >10    85 >10    86 n.d.  87n.d.  88 n.d.  89 n.d.  90 n.d.  91 n.d.  92 n.d.  93 n.d.  94 n.d.  95n.d.  96 n.d.  97 n.d.  98 n.d.  99 5.7 100 n.d. 101 3.3 102 n.d. 103n.d. 104 n.d. 105 n.d. 106 n.d. 107 n.d. 108 n.d. 109 1.5 110 1.5 1112.4 112 3.2 113 n.d. 114 n.d. 115 3.4 116 2.2 117 >10   118 4.0 119 n.d.120 n.d. 121 n.d. 122 8.0 123 n.d. 124 n.d. 125 n.d. 126 n.d. 127 n.d.128 n.d. 129 n.d. 130 n.d. 131 n.d. 132 n.d. 133 1.7 134 1.0 135 n.d.136 1.3 137 n.d. 138 n.d. 139 1.7 140 2.2 141 n.d. 142 n.d. 143 n.d. 144n.d. 145 n.d. 146 n.d. 147 n.d. 148 n.d. 149 n.d. 150 n.d. 151 n.d. 152n.d. 153 n.d. 154 n.d. 155 2.6 156 n.d. 157 9.2 158 n.d. 159 n.d. 160n.d. 161 n.d. 162 n.d. 163 n.d. 164 n.d. 165 n.d. 166 n.d. 167 n.d. 168n.d. 169 n.d. 170 n.d. 171 n.d. 172 n.d. 173 n.d. 174 n.d. 175 n.d. 176n.d. 177 n.d. 178 n.d. 179 n.d. 180 n.d. 181 n.d. 182 n.d. 183 n.d. 1846.5 185 n.d. 186 n.d. 187 n.d. 188 n.d. 189 n.d. 190 n.d. 191 n.d. 192n.d. 193 n.d. 194 n.d. 195 n.d. 196 n.d. 197 n.d. 198 1.3 199 3.2 2511.5 252 n.d. 253 n.d. 254 1.3 255 7.1 256 >10   257 >10   258 >10   259n.d. 260 n.d. 261 0.6 262 n.d. 263 >10   264 >10   265 >10   266 n.d.267 5.9 268 >10   269 0.8 270 0.8 271 0.8 272 8.1 273 >10   274 >10  275 >10   276 >10   277 n.d. 278 n.d. 279 n.d. 280 3.8 281 5.9 282 n.d.283 >10   284 n.d. 285 n.d. 286 n.d. 287 9.0 288 n.d. 289 n.d. 290 3.0291 n.d. 292 n.d. 293 n.d. 294 1.1 295 >10   296 n.d. 297 >10   298 n.d.299 n.d. 300 0.9 301 >10   302 n.d. 303 >10   304 >10   305 n.d. 306 1.0307 1.3 308 >10   309 2.0 310 n.d. 311 n.d. 312 7.7 313 7.8 314 n.d. 3155.7 316 n.d. 317 3.2 318 3.1 319 >10   320 1.0 321 n.d. 322 n.d. 323n.d. 324 n.d. 325 n.d. 326 n.d. 327 n.d. 328 n.d. 329 n.d. 330 n.d. 331n.d. 332 n.d. 333 n.d. 334 n.d. 335 n.d. n.d. means not determined

Example A12: YAP Nuclear Translocation Assay (JHH-5 Cells)

The YAP nuclear translocation assay is performed using compound-treatedJHH-5 cells (Fujise et al., Hepatogastroenterology. 1990 October;37(5):457-60) that are analyzed by high-content imaging following ananti-YAP1 antibody stain. Briefly, JHH-5 cells are suspended at0.48×10A6 cells/mi in William's E medium without phenol-red (ThermoFisher Scientific Gibco catalog number A1217601) supplemented with 10%(v/v) heat-inactivated fetal bovine serum (Thermo Fisher ScientificGibco catalog number 16140071) and penicillin-streptomycin at 100 U/mleach (Thermo Fisher Scientific Gibco catalog number 15140122). Cells aredispensed into 384 well clear bottom culture plates (50 ul/well, at24000 cells per well, Greiner catalog number 781091), and incubated for24 hours in a cell culture incubator at 37° C. Test compounds aretransferred into culture plates containing JHH-5 cells using an Echo550(Labcyte) acoustic dispenser (50 nl/well). Following an incubation of 4hours at 37° C., 12.5 ul of 20% PFA stock solution (Electron MicroscopySciences catalog number 15713S) is dispensed into each well for a finalconcentration of 4% (v/v) and incubated for 20 minutes at roomtemperature. Plates are washed with TBS (prepared from ten-foldconcentrate, Sigma Aldrich catalog number T5912) and then permeabilizedwith 0.1% (v/v) Triton X-100 solution (Sigma Aldrich catalog number93443) in DPBS for 15 minutes at room temperature. Plates are againwashed with TBS, followed by addition of anti-YAP1 antibody (NovusBiologicals catalog number NB110-58358) to each well at a dilution of1:500 in 3% (w/v) BSA in DPBS (prepared from 35% stock solution, SigmaAldrich catalog number A7979) and incubation overnight at 4° C. Afterremoval of primary antibody solution and washing with TBS, each well isincubated for 2 hours at room temperature with a 1:1000 dilution of goatanti-rabbit secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific catalog number A-21244) in 3% (w/v) BSA in DPBS(prepared from 35% stock solution, Sigma Aldrich catalog number A7979)supplemented with Hoechst 33342 solution (Thermo Fisher Scientificcatalog number 3570) at a 1:10000 dilution. Plates are again washed withTBS and then sealed and imaged on the IN Cell Analyzer High-ContentAnalysis System (GE Healthcare Life Sciences). By comparison of Hoechstnuclear stain to total intracellular YAP Alexa Flour 647 stain using theCellProfiler image analysis software (Carpenter et al., Genome Biol.2006; 7(10):R100), the signal corresponding to nuclear YAP is derived.Nuclear YAP signal x is normalized to active control and neutral controltreatment (DMSO), scoring median NC as 0% and median AC as +100% tocalculate normalized nuclear YAP signal xn per well as [xn=±100(x−NC)/(AC−NC)]. The control-normalized data were used for automatedcurve fitting to derive curve parameters per compound, including EC₅₀values.

Example A13: In Vivo Treatment of Mice

All animal studies were carried out in accordance with federal, state,local and institutional guidelines governing the use of laboratoryanimals in research. Male C57BL/6J mice (age 8-10 weeks) were purchasedfrom Jackson Labs (Bar Harbor, Me.). Example 46 was formulated for oraldosing at 3 mg/ml, and Example 261 at 1 mg/ml, in a suspension ofmethylcellulose:Tween80:Water (0.5:0.5:99). Single oral dose of eithercompound was at 10 mL/kg body weight. Blood and liver tissue werecollected under deep anesthesia (isoflurane) at either 2 h post-dose(for mRNA analysis) or 24 h post-dose (for immunohistochemistry).

Phospho-YAP (pYAP) HTRF in Liver Tissue

Frozen liver tissue was transferred into a Lysing Matrix tube (MP, cat#6913-500) with a 1:50 solution of Protease inhibitor (Sigma-Aldrich,cat #P8340) in Phosphosafe extraction buffer (Novagen, cat #71296-3) andhomogenized using the FastPrep-24 (MP biomedicals). The mixture was kepton ice for 20 min and then centrifuged at 14000 rpm for 20 min. Thesupernatant was transferred to a new tube. The protein concentration wasmeasured using the BCA Protein Assay Kit (Pierce cat. 23227) followingthe manufacturer's protocol. All samples were normalized to a finalconcentration of 5 μg/μl in a total volume of 180 μl of Phosphosafe andplated in a 96 well plate for the HTRF assay. pYAP was assayed by HTRFusing the Phospho-YAP (SER127) 50000 test kit (Cisbio, catalogue number64YAPPEI). Mouse liver lysates in extraction buffer alone (Phosphosafe)or 1:1 mix of Phosphosafe with HTRF Lysis buffer (Cisbio) were assayedin duplicate wells of 384-well assay plates (12 μL/well). The same inputquantity was used for each sample, based on total protein content (30-60ug per sample per well). Detection antibodies phospho-YAP d2 andphospho-YAP cryptate each diluted 1:40 in Detection buffer (Cisbio) wereadded (3 μL/well, final dilution 1:200 for each antibody) after whichthe plates were sealed and incubated overnight (18 hrs) at roomtemperature protected from light. Plates were read on an EnVision(PerkinElmer) with emission wavelengths of 665 nm & 620 nm. The HTRFsignal ratio was calculated as Signal(665 nm)/Signal(620 nm))×10000 asspecified in the manufacturer's protocol. One HTRF signal value (mean ofduplicate wells) was used for each lysate sample in treatment groupplots.

Quantitative RT-PCR

Total liver RNA was isolated from tissue after storage in RNAlaterreagent (Qiagen), using a TRIzol method according to the manufacturer'sinstructions (Life Technologies). Relative mRNA abundance was measuredby quantitative RT-PCR using TaqMan gene expression assays and reagentsaccording to standard protocols (Applied Biosystems). Mouse probes forYAP target genes were CTGF (Mm01192933_g1) and Cyr61 (Mm00487498_m1).Mouse GAPDH was used as an internal control (probe 4351309). Relativechanges in mRNA were calculated by the AACt method. Changes in geneexpression with compound treatment are expressed as fold-change relativeto the vehicle group.

Immunohistochemistry (IHC) for Ki67

Liver tissue was fixed in 10% neutral buffered formalin for 24-48 h.Processing and embedding in paraffin was by standard procedures.Sections were cut at 5 microns thickness and stained using a Ventanaautomated system. Primary antibody for mouse Ki67 was a rabbit IgG(Bethyl Labs). Ki67-positive hepatocytes were quantified by imageanalysis using the HALO platform. At least 3 sections per animal wereanalyzed, from the middle of the medial lobe.

TABLE 1E Results of compound treatment in mouse pYAP (% of CTGF mRNACyr61 mRNA Ki67 IHC control) (fold-change) (fold-change) (fold-change)Example 46 64 2.1 (+/−0.4) 3.5 (+/−0.5) 3.3 (+/−0.2) 30 mg/kg Example261 60 2.5 (+/−0.2) 3.9 (+/−0.6) 3.1 (+/−0.5) 10 mg/kg

mRNAs for YAP target genes CTGF and Cyr61 were increased 2 h andproliferation indicated by Ki67 immunohistochemistry (IHC) was increased24 h post single oral dose of LATS kinase inhibitors. Values areexpressed as fold-change relative to the vehicle group with(mean+/−SEM).

Example B1: Human limbal epithelial cell isolation Research-consentedcadaveric human corneas were obtained from eye banks. Limbal rims weredissected and partially dissociated in a 1.2 mg/ml dispase solution for2 hours at 37° C. followed by 10 minutes in TrypLE (Life Technologies).Pieces of limbal crypts were then carefully cut out of the partiallydissociated limbal rims and rinsed by centrifugation). Cells obtained inthis manner were used in the Examples B2-B111 below.

Example B2: Exposure of Cells to LATS Inhibitors and Measurement ofIntracellular YAP Distribution

Cells obtained as described in Example 1 were plated in glass-bottomblack wall 24-well dishes in limbal epithelium cell culture medium (DMEMF12 supplemented with 10% human serum and 1.3 mM calcium chloride)supplemented with LATS inhibitor compound example no. 133 or 49 at aconcentration of 10 micromolar or supplemented in DMSO as a negativecontrol. Cells were cultured under these conditions for 24 hours at 37°C. in 5% CO2.

To measure the effect of the LATS inhibitors on the downstream targetYAP, intracellular YAP distribution was analyzed byimmunohistochemistry. Cell cultures were fixed with 4% PFA for 20minutes, permeabilized and blocked in a blocking solution of 0.3% TritonX-100 (Sigma-Aldrich) and 3% donkey serum in PBS for 30 minutes. Cellswere then labeled with primary antibody in the blocking solution for 12hours at 4° C. Primary antibody used was anti-YAP from Santa CruzBiotechnology. Samples were washed in PBS three times and donkey-raisedsecondary antibody Alexa Fluor 488 (Molecular Probes) at 1:500 dilutionwere applied for 30 minutes at room temperature. Negative control wasomitted primary antibody (data not shown). Fluorescence was observedusing a Zeiss LSM 880 confocal microscope.

Only weak YAP immunostaining was observed in the nucleus of LSCscultured without the LATS inhibitors (DMSO control). YAP immunostainingwas stronger in the nucleus of LSCs exposed to the LATS inhibitorcompound example no. 133 or 49 (data not shown).

Example B3: Exposure of Cells to LATS Inhibitors and Measurement of YAPPhosphorylation

Cells obtained as described in Example B1 were detached from the culturedish with Accutase for 10 minutes at 37° C., cell suspensions wererinsed by centrifugation and plated in DMEM F12 supplemented with 10%human serum and 1.3 mM calcium chloride in 6-well plates (Corning) andcultured without LATS inhibitor compounds for 2-4 days.

The medium was then replaced by fresh limbal epithelium cell culturemedium (DMEM F12 supplemented with 10% human serum and 1.3 mM calciumchloride) supplemented with LATS inhibitor compound example no. 133 or49 at a concentration of 10 micromolar or supplemented in DMSO as anegative control. Cells were cultured under these conditions for 1 hourat 37° C. in 5% CO2.

To measure the effect of the LATS inhibitors on the downstream targetYAP, the YAP phosphorylation levels were measured by western blot asfollows. The cell pellets were obtained by trypsin dissociation andcentrifugation and washed with PBS. The pellets were lysed with 30microlitres of RIPA lysis buffer containing protease inhibitor cocktail(Life Technologies) for 30 minutes, with vortexing every 10 minutes. Thecell debris were then pelleted at 4° C. for 15 minutes at 14 k rpm andthe protein lysate was collected. Protein concentration was quantifiedusing a micro BCA kit (Pierce). Fifteen micrograms of total protein wasloaded in each well of 4-20% TGX gels (BioRad) and Western blotting wasperformed according to the manufacturer's instructions. Membranes wereprobed with phospho-YAP (ser127) (CST, 1:500) or total Yap (Abnova,1:500) antibody and actin (Abcam) labelling was used as loading control.Membranes were stained with HRP-conjugated secondary antibodies, rinsedand imaged using a ChemiDoc system (Biorad) according to themanufacturer's instructions.

Western blot analysis (see FIG. 1 ) showed that both compound exampleno. 133 and 49 caused a reduction in YAP phosphorylation levels in humanLSCs. These results suggest that the LATS inhibitor compound example no.133 and 49 can activate YAP signaling in human LSCs.

Example B4: Human Limbal Stem Cell Population Expansion andImmunohistochemical Observation of Cellular Phenotype

Cells obtained as described in Example B1 were plated in 24-well plates(Corning) in limbal epithelium cell culture medium (DMEM F12supplemented with 10% human serum and 1.3 mM calcium chloride)supplemented with LATS inhibitor compound example no. 133 or 49 at aconcentration of 10 micromolar or supplemented in DMSO as a negativecontrol. Cells were first cultured at 37° C. in 5% CO2 for 6 days afterisolation without passaging (FIGS. 2A, 2B and 2C).

To evaluate the ability of the compounds to enable LSC expansion aftertwo passages, LSCs were passaged and cultured for two weeks in thepresence of compound example 49 to enable expansion (FIG. 2D). Limbalstem cells (LSCs) were passaged by treating cultures with Accutase for10 minutes at 37° C., rinsing the cell suspension by centrifugation andplating cells in fresh LSC culture medium supplemented with LATSinhibitor compound example 49.

In order to observe that the expanded cell population expressedp63alpha, this was measured by immunohistochemistry as follows. Cellcultures were fixed with 4% PFA for 20 minutes, permeabilized andblocked in a blocking solution of 0.3% Triton X-100 (Sigma-Aldrich) and3% donkey serum in PBS for 30 minutes. Cells were then labeled withprimary antibody in the blocking solution for 12 hours at 4° C. Primaryantibody used was p63alpha from Cell Signalling. Samples were washed inPBS three times and donkey-raised secondary antibody Alexa Fluor 488(Molecular Probes) at 1:500 dilution were applied for 30 minutes at roomtemperature. Cells were counter-stained with a human nuclear antigenantibody (Millipore) at a 1:500 dilution in order to label all cells inthe culture and confirm their human identity. Negative control wasomitted primary antibody (data not shown). Fluorescence was observedusing a Zeiss LSM 880 confocal microscope.

FIG. 2A shows that in the presence of growth medium and DMSO, only a fewisolated cells attach to the culture dish and survive up to 6 days. Mostcells expressed the human nuclear marker, but few expressed p63alpha. Incontrast, in the presence of LATS inhibitors compound example no. 133(FIG. 2B) and compound example no. 49 (FIG. 2C), the cells formedcolonies and expressed p63alpha. This result indicated that the LATSinhibitors promote the expansion of the population of cells with thep63alpha-positive phenotype. FIG. 2D: Passaging cells and culturing themin the presence of LATS inhibitor compound example no. 49 for two weeksenabled cell population expansion and the formation of confluentcultures expressing p63alpha.

Example B5: Human Limbal Stem Cell Population Expansion and MeasurementThereof

Cells obtained as described in Example B1 were plated in 48-well plates(Corning) in XVIVO15 medium (Lonza) supplemented with LATS inhibitors(as listed in Table 2 and 3 below) at a concentration of 10 micromolaror supplemented in DMSO as a negative control. Cells were cultured at37° C. in 5% CO2.

For each compound, two sets of cultures were generated. A first set ofcultures was fixed in 4% PFA for 20 minutes at room temperature aftercells isolated from the cornea had attached to the cell culture dish(typically 24 h after cell plating). A second set of cultures was fixedin 4% PFA for 20 minutes at room temperature after being cultured fortwo passages. Cells were passaged when they reached 90-100% confluence.

In order to observe that the expanded cell population expressedp63alpha, this was measured by immunohistochemistry as follows. Thefixed cell cultures were permeabilized and blocked in a blockingsolution of 0.3% Triton X-100 (Sigma-Aldrich) and 3% donkey serum in PBSfor 30 minutes. Cells were then labeled with primary antibody in theblocking solution for 12 hours at 4° C. Primary antibody used wasp63alpha from Cell Signalling. Samples were washed in PBS three timesand donkey-raised secondary antibody Alexa Fluor 488 (Molecular Probes)at 1:500 dilution were applied for 30 minutes at room temperature. Cellnuclei were then labeled in a solution of 0.5 micromolar of Sytox Orange(ThermoFisher) in PBS for 5 minutes at room temperature.

To evaluate the percentage of p63alpha-positive cells, the number ofcells labeled by the anti-p63alpha antibody was counted and the totalnumber of cells was determined by counting the number of nuclei stainedby Sytox Orange. The proportion of p63alpha-positive cells was thendetermined by calculating the percentage of Sytox-orange-positive nucleithat also expressed p63alpha.

To evaluate cell expansion ratios, nuclei were counted using a Zeiss LSM880 confocal microscope. The expansion factor was then determined bycalculating the ratio of the expanded population of cells to populationof seeded cells.

Results in the Tables below indicate that the LATS inhibitors enabledcell population expansion. In the presence of the LATS inhibitors, 57 to97 percent of the cells express the p63alpha-positive phenotype.

TABLE 2 Compound Example No. Expansion factor Ex. 47 2137 Ex. 12 2087Ex. 49 2029 Ex. 261 1717 Ex. 62 1712 Ex. 14 1423 Ex. 6 1275 Ex. 288 1241Ex. 133 1205 Ex. 66 1160 Ex. 290 1051 Ex. 65 1048 Ex. 287 991 Ex. 17 976Ex. 139 961 Ex. 11 705 Ex. 289 681 Ex. 33 39 DMSO 35

TABLE 3 Percentage of Percentage of Compound p63a-positive Compoundp63a-positive Example No. cells Example No. cells Ex. 47 97 Ex. 290 90Ex. 12 95 Ex. 65 87 Ex. 49 92 Ex. 287 86 Ex. 261 93 Ex. 17 86 Ex. 62 95Ex. 139 87 Ex. 14 93 Ex. 11 86 Ex. 6 93 Ex. 289 80 Ex. 288 95 Ex. 33 6Ex. 133 89 DMSO 3 Ex. 66 89

Example B6: siRNA Knockdown of LATS1 and LATS2 in Human Limbal Cells

Cells obtained as described in Example 1 were plated in 24-well plates(Corning) in XVIVO15 medium (Lonza). Cells were cultured at 37° C. in 5%CO2. LATS1 and LATS2 were knocked down by transfection (lipofection,using RNAiMax, Thermofisher). Each well of the cell culture plate wastransfected with 0.5 micrograms of pools of 4 siRNAs targeting eachgene. siRNAs used in this study were Qiagen's LATS1 siRNA S100067172 andLATS2 siRNA S100106925. Scrambled siRNAs were used as negative controlsaccording to the manufacturer's protocol (Qiagen).

In order to measure cell proliferation, EdU staining was performedaccording to the manufacturer's instructions (Life Technologies) 48hours after transfection with LATS1 and LATS2 siRNAs or scrambled siRNAcontrols. Cell nuclei were labeled with Sytox Orange. EdU and SytoxOrange fluorescence was observed using a Zeiss LSM 880 confocalmicroscope in order to measure the percentage of EdU-positive cellnuclei.

FIG. 3 shows that the percentage of EdU-positive cells increased uponLATS1 and LATS2 knockdown, showing that knockdown of LATS leads to cellproliferation.

Example B7: Human Limbal Stem Cell Population Expansion andImmunohistochemical Observation of Markers DeltaN-p63 Alpha/Beta/Gamma,ABCG2 and C/EBP delta

Cells obtained as described in Example 1 were plated in 48-well plates(Corning) in XVIV015 medium (Lonza) supplemented with LATS inhibitors(Compound ex. 49 and compound ex. 133) at a concentration of 10micromolar or supplemented in DMSO as a negative control. Cells werecultured at 37° C. in 5% CO2 for 8 to 10 days.

In order to observe that the expanded cell population expresses markersnormally expressed by limbal stem cells, the ability of the cells toexpress DeltaN-p63 alpha/beta/gamma, ABCG2 and C/EBP delta was measuredby immunohistochemistry as follows. Cell cultures were fixed with 4% PFAfor 20 minutes, permeabilized and blocked in a blocking solution of 0.3%Triton X-100 (Sigma-Aldrich) and 3% donkey serum in PBS for 30 minutes.Cells were then labeled with primary antibody in the blocking solutionfor 12 hours at 4° C. Primary antibodies used were p63a (Cell SignalingTechnology), ABCG2 (EMD Millipore), and C/EBP6 (Abcam), cytokeratin 15,cytokeratin 12 (Abcam). Samples were washed in PBS three times anddonkey-raised secondary antibody Alexa Fluor 488 (Molecular Probes) at1:500 dilution were applied for 30 minutes at room temperature. Cellnuclei were then labeled in a solution of 0.5 micromolar of Sytox Orange(ThermoFisher) in PBS for 5 minutes at room temperature. Samples werethen observed using a Zeiss LSM 880 confocal microscope.

Results indicated (see FIG. 4 ) that cells cultured in XVIVO mediumsupplemented with DMSO alone (no LATS inhibitor compound) do nottypically express markers normally expressed by LSCs. In contrast, cellscultured in the presence of LATS inhibitors express markers normallyexpressed by LSCs.

Example B8: Human Limbal Stem Cell Differentiation into CornealEpithelial Cells

Cells obtained as described in Example 1 were plated in 48-well plates(Corning) in XVIV015 medium supplemented with LATS inhibitors compoundexample no. 49, 47, 12 or 261 at a concentration of 10 micromolar orsupplemented in DMSO as a negative control. Cells were cultured at 37°C. in 5% CO2 for 8 to 10 days.

Analysis was then conducted to determine whether a population ofp63alpha-positive LSCs expanded in the presence of LATS inhibitorsretained the ability to differentiate into keratin-12-positive cornealepithelial cells, which is required for corneal clarity to be restoredin the patients that receive the LSC transplant.

To promote LSC differentiation into corneal epithelium cells, theculture medium was changed to DMEM (Invitrogen) without serum or LATSinhibitors and cells were cultured for 4-8 days. In order to observe LSCdifferentiation into corneal epithelium cells, the cell cultures werefixed with 4% PFA for 20 minutes, permeabilized and blocked in ablocking solution of 0.3% Triton X-100 (Sigma-Aldrich) and 3% donkeyserum in PBS for 30 minutes. Cells were then labeled with primaryantibody in the blocking solution for 12 hours at 4° C. Primaryantibodies used were p63a (Cell Signaling Technology) and cytokeratin 12(Abcam). Samples were washed in PBS three times and donkey-raisedsecondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 647(Molecular Probes) at 1:500 dilution were applied for 30 minutes at roomtemperature. Samples were then observed using a Zeiss LSM 880 confocalmicroscope.

Results indicated that cultures maintained in the presence of LATSinhibitors and then induced to differentiate, contained areas whereclusters of p63alpha-positive cells are contiguous to clusters of cellsthat are p63alpha-negative, but keratin-12-positive (FIG. 5 ). Thereforelimbal cells expanded in the presence of LATS inhibitors candifferentiate into keratin-12-positive cells when under appropriateconditions.

Example B9: Delivery of LSC-Biomatrix Preparation to Rabbit Eyes RabbitModel of Limbal Stem Cell Deficiency

Limbal stem cell deficiency (LSCD) was unilaterally created in the righteye of NZA rabbits by debridement of epithelial cells and ablation oflimbal stem cells (LSC) with Whatman paper soaked with a solution of 1Mof sodium hydroxide. Limbal conjunctiva was treated with Mitomycin C toreduce corneal neovascularization. The left eye was left intact. Thecell population expanded in LATS inhibitor compound example no. 12 wasdelivered as described below. After cell delivery, rabbits receivedanalgesic treatment (Tramadol 10 mg/kg PO BID in first 2 weeks,Meloxicam 0.3 mg/kg PO SID in first 2 weeks or as long as the animalshowed signs of ocular discomfort), anti-inflammatory treatment (Ancef®(cefazolin) 50 mg sub-Tenon immediate post-procedure, Tobrex® ophthalmicsolution t.i.d in 1st week and b.i.d thereafter, ampicillin 80 mg/kg/day(40 mg BID) SQ for first week) and immunosuppression (Cyclosporine A(0.5%) top oc. t.i.d 1st week and b.i.d. thereafter, Gentocin®-Durafilm®(Gentamicin sulfate and betamethasone, MERCK) top oc. t.i.d 1st week andb.i.d. thereafter, Cyclosporine A (5 mg/kg/day) SQ in 1st week and then% dosage thereafter).

Biomatrix Preparation

Gelatin methacrylate (GelMA) was synthesized according to a previouslypublished protocol. (Nichol, J. W. et al. Cell-laden microengineeredgelatin methacrylate hydrogels. Biomaterials 31, 5536-5544,doi:10.1016/j.biomaterials.2010.03.064 (2010). In brief, 20 grams ofporcine derived gelatin (Cat #G2500, Sigma) was dissolved overnight at50° C. in 200 ml of PBS without calcium and magnesium (DPBS, Cat#21-031, Corning). With strong agitation, methacrylic anhydride (Cat#276685, Sigma) was added dropwise (approximately 1 ml/min) into thegelatin solution to reach the concentration of 8% (vol/vol). The mixturewas stirred at 60° C. in an oil bath for 3 hours before adding 200 ml ofthe DPBS and followed by thorough mixing for additional 15 minutes. Thediluted mixture was purified via dialysis against Milli-Q water usingdialysis tubings (15 kDa MWCO, Spetrua/Por) for 1 week at 45° C. toremove methacrylic acid. The purified samples were lyophilized andstored at −80° C. until further use.

To prepare a 15% GelMA stock solution, 1.5 gram of the freeze-driedGelMA foam was dissolved in 10 ml of pre-warmed DPBS at 37° C. After theGelMA foam was fully dissolved, the photoinitiator was introduced byadding into the GelMA solution 15 mg of lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP). 500 microlitre of 1NNaOH (Cat #BDH-7222-1, VWR) was added to the solution to adjust the pHto neutral before the solution was filtered using 0.22 micrometresterile membranes (Millipore). The final filtrate was separated into 500microlitre aliquots and stored at 4° C. until further use.

LSC Transplantation In Vivo.

Cells obtained as described in Example 1 were plated in 6-well plates(Corning) in XVIV015 medium supplemented with LATS inhibitor compoundexample no 12 at a concentration of 10 micromolar. Cells were culturedat 37° C. in 5% CO2 for two passges (two weeks).

Cell suspensions for transplantation were prepared as follows. Cellswere detached from the culture dish with Accutase for 10 minutes at 37°C. Cells were then rinsed by centrifugation and resuspended in 16microlitres of XVIV015 medium without LATS inhibitors.

A 5% GelMA solution was prepared by mixing 16 microlitres of the LSCsuspension or saline control with 8 microlitres of the 15% GelMA stocksolution and followed by adding all the mixture to the therapeuticcontact lens (Lotrafilcon B, Alcon) on a supporting glass slide. Thefinal solution contained 300000 cells and 5% GelMA. The power of the UVLED light source (365 nm, Hamamatsu) was adjusted to 15% with a UVintensity reading of 30 mW/cm2. A brief UVA exposure of 25 seconds wasused for the photopolymerization process to bioprint the human LSCs tothe inner surface of the contact lens. After the polymerization process,the LSC loaded contact lens was applied to a dissected rabbit eye exvivo or to the rabbit eye in vivo.

For experiments where the LSC-loaded contact lens was applied ex vivo,samples were observed under a Zeiss epifluorescence microscope withintwo hours.

For in vivo experiments, the LSC-loaded contact lens was applied to theright eye of the rabbit model of LSCD described above and tarsorrhaphywas performed to keep the contact lens on the ocular surface for 1 week.After one week, the contact lens was removed and rabbits were kept alivefor an additional 4 weeks.

Rabbits were sacrificed five weeks after cell delivery, corneas weredissected and fixed in 4% PFA. Samples were embedded in paraffin andsectioned. Immunohistochemistry was performed to detect the presence ofkeratin-12, a marker of corneal epithelium cells and keratin-19, amarker of conjunctival cells. The anti-keratin 12 and 19 primaryantibodies were from Abcam. Human mitochondrial protein was used toconfirm the human origin of the cells. The anti-human mitochondrialprotein primary antibody was from Novus. Samples were then stained withHRP-conjugated secondary antibodies (ThermoFisher) and observed under aZeiss microscope.

Results indicated that LSCs attached to a contact lens using GelMApolymerization can be delivered to the surface of the rabbit eye ex vivo(FIG. 6 ).

FIG. 7 shows that in a rabbit model of limbal stem cell deficiency, aLSC population expanded in the presence of the LATS inhibitor (compoundex. 12), attached to a contact lens using GelMA polymerization anddelivered in vivo to the rabbit's corneal surface, lead to regenerationof the a keratin-12-positive corneal epithelium and preventedconjunctivalization by keratin-19-positive conjunctival cells in thetransplanted eye (FIGS. 7A and B respectively). In contrast,non-transplanted rabbit eyes showed absence of keratin-12-positivecorneal epithelium restoration. Instead, signs of conjunctivalizationwere observed, as showed by the presence of keratin-19 staining (FIGS.7C and D respectively).

FIG. 8 shows that the cells that restored the corneal epithelium oftransplanted eyes were human, as demonstrated by the presence of humanmitochondrial protein (FIG. 8A). In contrast, the ocular surface ofnon-transplanted eyes did not exhibit human mitochondrial proteinstaining (FIG. 8B).

Example B10: Delivery of LSC Ex Vivo Using TISSEEL

To prepare TISSEEL (Baxter, cat #NDC 0944-4301-02) working solution,manufacturer's instructions were followed to reconstitute the suppliedingredients. Further dilution of the reconstituted fibrinogen solutioncan be done by adding sterile water to reach desired concentration offibrinogen. Thrombin working solution was diluted 500 times withDulbecco's phosphate-buffered saline (DPBS, GIBCO, Thermo FisherScientific, Waltham, Mass.) to achieve 1 unit/ml solution.

LSC Delivery to Culture Surface In Vitro.

Cells obtained as described in Example B1 were plated in 6-well plates(Corning) in XVIVO15 medium supplemented with LATS inhibitor compoundexample no 12 at a concentration of 10 micromolar. Cells were culturedat 37° C. in 5% CO₂ for two passages (two weeks).

Cell suspensions for transplantation were prepared as follows. Cellswere detached from the culture dish with Accutase for 10 minutes at 37°C. Cells were then rinsed by centrifugation and resuspended in thethrombin working solution mentioned above and the final cell densitieswere adjusted by serial dilution to between 0.1 million per ml and 30million per ml.

Immediately after mixing the cell suspension with fibrinogen solution,10 ul of the mixture were plated to the center of a collagen coated 24well plate and incubated at room temperature for 5 minutes to solidifythe fibrin. After replenishing each well with XVIV015 medium withoutLATS inhibitors, all the samples were monitored for 2 weeks for fibrindegradation and cell repopulation. The results (FIG. 9 ) suggested thatTISSEEL was used successfully to deliver LSCs, and the culture area,similar size to human cornea, can be covered between 1 week and 2 weeksin the samples with 100 k and 300 k LSCs delivered initially.

LSC Delivery to Human Cornea Ex Vivo.

Cells obtained as described in Example 1 were plated in 6-well plates(Corning) in XVIVO15 medium supplemented with LATS inhibitor compoundexample no 12 at a concentration of 10 micromolar. Cells were culturedat 37° C. in 5% CO₂ for two passages (two weeks). One day before thedelivery experiment, cells were labeled with CellTracker green CMFDA dye(Thermo Fisher Scientific, Waltham, Mass.) according to themanufacturer's protocol.

Cell suspensions for transplantation were prepared as follows. Cellswere detached from the culture dish with Accutase for 10 minutes at 37°C. Cells were then rinsed by centrifugation and resuspended in thethrombin working solution mentioned above to reach the final celldensity of 30 million per ml.

Immediately after mixing the cell suspension with fibrinogen solution,10 ul of the mixture were plated to the center of a human cornea andincubated at room temperature for 5 minutes to solidify the fibrin. ALotraB contact lens (Air Optix Night and Day, Alcon) was used to coverthe LSC-fibrin construct on the human cornea to mimic the real clinicalprocedure. Fluorescence images were taken by a Nikon fluorescencestereomicroscope. The data (FIG. 10 ) demonstrated that TISSEEL wassuccessfully used to deliver high number of LSCs onto corneal surface exvivo and the LSC-fibrin construct appears robust to tolerate furthermanipulation such as applying protective contact lens.

Example B11: Delivery of Multiple Cell Populations to Ocular Surfaceinto Designated Patterns Via Bioprinting

A customized bioprinter was built based on DOPsL technology as publishedin Advanced Materials (2015, DOI: 10.1002/adma.201202024).

Cell suspensions for bioprinting were prepared as follows. HEK-293 cellswere pre-labelled with Celltracker green CMFDA dye or red CMTPX dye(Thermo Fisher Scientific, Waltham, Mass.) before harvest. 25 μl of thecell suspension was mixed with 25 μl of the 15% GelMA stock solution toreach a final cell density of 200 million cells/ml.

Rabbit eye balls were placed onto a petri dish filled with 1% lowmelting point agarose and the epithelium was carefully debrided by asurgical scalpel immediately before the bioprinting process. Thecell-GelMA mixture was then applied to the top of the cornea with aPolydimethylsiloxane (PDMS) o-ring (Specialty Silicone Products, Inc.,Ballston Spa, NY) placed on the limbus to prevent the cell-GelMA mixturedripping from the curved ocular surface.

A customized light pattern (i.e. half of the Yin-Yang pattern) wasprojected to the cell(green)-GelMA mixture on top of the rabbit eye for4 seconds before the unpolymerized mixture was rinsed away. Anotherlight pattern (i.e. the other half of the Yin-Yang pattern) wasprojected to the sample for 4 seconds after a new cell (red)-GelMAmixture was applied to the top of the same rabbit cornea. Several rinseswith PBS were performed before imaging. See FIG. 11 .

Example B12: Reducing Immune Rejection by CRISPR/Cas9-Mediated Deletionof the Beta-2-Microglobulin Gene in LSCs

In the example below, HLA class I expression was eliminated from the LSCsurface by CRISPR-mediated deletion of the beta-2-microglobulin gene.

Cell transfection: LSCs obtained as described in Example B1 werecultured in X-VIVO15 medium supplemented with the LATS inhibitorcompound example no 48a to a confluency of 50-60% in a 35 mm petri disheight days after LSC isolation. LSCs were transfected with a mixture oftracrRNA-crRNA-Cas9 mRNA. To obtain the mixture for the size of a 35 mmpetri dish, 12.5 microlitre of 10 micromolar tracrRNA (Dharmacon, Cat#U-002000-20), 12.5 microlitre of 10 micromolar crRNA targeting humanB2M (Dharmacon, Cat #CR-004366-01), 50 microlitre of 0.1microgram/microlitre Cas9 mRNA (Dharmacon, Cat #CAS11195), and 15microlitre of DharmaFECT Duo Transfection Reagent (Dharmacon, Cat#T-2010-02) were combined and incubated for 20 minutes at roomtemperature. The mixture was added drop wise to the culture dish in 2.5ml the medium (XVIVO15 supplemented with the LATS inhibitor compoundexample no 48a) (w/o antibiotics). In order to reduce mRNA cytotoxicity,0.2 microgram/ml B18R (eBioscience, Cat #34-8185-81) was added to themedium. The transfection reagent alone represents the transfectionnegative control.

After 6 h incubation in 5% CO₂ at 37° C. medium was replaced with freshX-VIVO15 medium supplemented with compound example no 48a (w/oantibiotics) including compound and B18R. After 72 h in a 5% CO2incubator cells were passaged 1:2 and expanded for additional 72 h inLSC medium (w/o antibiotics) including compound and B18R on synthemaxcoated petri dishes to obtain more cells for FACS sorting.

FACS analysis: LSCs were treated with Accutase (ThermoFisher, Cat #A1110501) for 20 minutes in 5% CO2 at 37° C. After scraping the cells, thereaction was stopped by using cell culture medium containing 10% Serumand transferred to a falcon tube for a centrifugation step (1000 rpm, 5minutes). After aspirating the medium cells were resuspended in 200microlitre FACS buffer (PBS/10% FBS).

To analyze the expression of B2M and HLA-ABC, 5 microlitre APC mouseanti-human P2-microglobulin antibody (Biolegend, Cat #316312) and 20microlitre PE mouse anti-human HLA-ABC antibody (BD Bioscience, Cat#560168) were added to the cell suspension and incubated for 30 minuteson ice. Cells were washed 3 times after antibody labelling with FACSbuffer and resuspended in 500 microlitres in FACS buffer. Before FACSsorting, cells were filtered through a 70 micrometre filter and storedon ice until sorting.

In order to prevent cells sticking to the wall, collection tubes werefilled with the serum for 30 minutes before the sort. Cells were sortedon a BD FACSAria II instrument into prepared collection tubes, usinghuman serum enriched LSC medium including compound and B18R. FACS datawere analyzed using BD FACSDiva software.

Results presented in FIG. 12 show that CRISPR-mediated deletion of B2Mand subsequent elimination of HLA A, B and C occurred in 21 percent ofthe LSCs. As the XVIV015 medium supplemented with the LATS inhibitorcompound example no 48a enables efficient expansion of LSCs, the 21percent B2M-negative/HLA A,B,C-negative population of LSCs could then beexpanded to produce a cell preparation where 97 percent of the cells donot express HLA class-I drivers of immune rejection (FIG. 13 ).

Example B13: Residual Compound Estimation Study Standard CurvePreparation

A dilution series of stock standards were made at concentrations of 100micromolar, 10 micromolar, 1 micromolar, 500 nM, 100 nM, and 0micromolar. Compound example no. 48a (10 mM in DMSO) stock was spikedinto 50% acetonitrile 50% water to make the spike standards. The spikingstandards were used to spike the blank media samples. 10 microlitres ofspiking standard was added to 90 microlitres of media to create thespiked media standards. 10,000 nM, 1000 nM, 100 nM, 50 nM, 10 nM, and 0nM media standards were used to generate the compound example no. 48astandard curve.

50 microlitres of each media sample was treated with 400 microlitres ofthe extraction solution. The extraction solution consists ofacetonitrile/methanol (3:1). The standard curve samples were extractedusing the same volumes and conditions as the unknown media samples. Theextracted samples were centrifuged at 10,000 rpm for 5 min. Aftercentrifugation, 200 microlitres of each sample supernatent wastransferred to a clean 96-well plate. The extracted samples wereanalyzed by High Resolution LC-MS. Thermo Xcalibur Software and QuanBrowser were used to generate the standard curve and calculate theconcentration values. An external calibration method was used tocalculate the concentration of compound example no. 48a in the culturemedia samples.

LC-MS Analysis

High-resolution chromatography was performed using the Thermo UltimateUPLC and a Kinetex 2.1×50 mM C18 RP column (2.6 micron particles). Themobile phases consisted of 5 mM Ammonium Acetate in H2O for Buffer A,and 0.1% Formic Acid in Acetonitrile for Buffer B. A standard binarylinear gradient was used for Reverse Phase Chromatography. The Thermo QExactive mass spectrometer was used for this study. The UPLC effluentwas directed into the Q Exactive Mass Spectrometer which was equippedwith an ESI ion source. The mass spectrometer was programmed to performin High Resolution Full Scan mode and MS2 mode.

Test System

Cell preparation for the LSC washout: the washout experiment wasperformed in triplicates, where each sample contained 0.5 million cells.All samples (media only, media with cells, supplemented with compoundexample no. 48a and cells in media supplemented with compound exampleno. 48a) were cultured at 37 degree incubator for the duration of theexperiment. The medium used for the experiment was X-VIVO 15.

The cells were cultured to passage 2 in X-VIVO-15 medium supplementedwith 3 micromolar compound example no. 48a. When the culture reached 70%confluency, the the medium was removed by aspiration and replaced withfresh medium supplemented with 3 micromolar compound example no. 48a;the cells were cultured for an additional six days. On day seven, thefollowing samples were generated:

-   -   1. 2 ml of “blank” medium (not supplemented with the compound)    -   2. 2 ml of medium cultured in the presence of the compound        (pre-wash)    -   3. 2 ml of media for each wash sample 1-10    -   4. Cell pellets (post-wash)

The cells were collected by using the sell lifter (Costar, cat #3008).

The media and cell pellet samples were analyzed and quantified usingHigh Resolution LC-MS.

To estimate the residual levels of compound example no. 48a, cellpellets and supernatent were collected. The amount of compound exampleno. 48a was determined by LC-MS. Estimation of compound example no. 48aconcentrations in the Pre-wash Media, Wash Media (Table 4) and the CellPellet are summarized (Table 5). Pre-wash Media samples have the highestlevels of compound example no. 48a (approx. 9,830 nM to 10,837 nMrange). Post-wash Pellet levels have low, but detectable levels ofcompound example no. 48a (nanomolar).

TABLE 4 Estimation of concentrations for compound example no. 48a in thePre-wash Media and Wash Media compound compound compound example no.example no. example no. 48a (nM) 48a (nM) 48a (nM) Volume in Triplicatein Triplicate in Triplicate Sample (mL) #1 #2 #3 Pre-wash Media 29829.547 10374.463 10837.706 Wash Media 1 2 461.271 401.058 354.547 WashMedia 2 2 31.933 38.721 18.400 Wash Media 3 2 BLQ BLQ BLQ Wash Media 4 2BLQ BLQ BLQ Wash Media 5 2 BLQ BLQ BLQ Wash Media 6 2 BLQ BLQ BLQ WashMedia 7 2 BLQ BLQ BLQ Wash Media 8 2 BLQ BLQ BLQ Wash Media 9 2 BLQ BLQBLQ Wash Media 10 2 BLQ BLQ BLQ BLQ = Beneath Limit of Quantification

TABLE 5 Estimation of concentrations for compound example no. 48a in theCell Pellet compound compound Avg of compound Total compound example no.48a example no. 48a example no. 48a # of cells example no. 48a Sample(nM) (pg/cell) (pg/cell) in Pellet (pg) PostWash_Pellet 1 20.342 0.000680.00068 0.5 × 10⁶ 340 PostWash_Pellet 2 19.908 0.00066 PostWash_Pellet 320.787 0.00069

The amount of compound example no. 48a in the Pre-wash Media from threeincubations ranges from 9,830-10,838 nM. The concentration of compoundexample no. 48a in the Post-wash Cell Pellet was in the range of19.9-20.8 nM.

Residual Amount of Other Compound Examples after Wash-Out

For compound example numbers 12, 261, and 5, residual amounts inexpanded LSCs were measured using the same method as for compoundexample no. 48a.

TABLE 6 Estimation of concentrations for compound ex. 12 in the CellPellet compound compound Avg of compound Total compound ex. 12 ex. 12ex. 12 # of cells ex. 12 Sample (nM) (pg/cell) (pg/cell) in Pellet (pg)PostWash_Pellet 1 7.370 0.000136 0.000178 1 × 10⁶ 178 PostWash_Pellet 214.443 0.000264 PostWash_Pellet 3 7.319 0.000134

TABLE 7 Estimation of concentrations for compound example no. 261 in theCell Pellet compound compound Avg of compound Total compound example no.261 example no. 261 example no. 261 # of cells example no. 261 Sample(nM) (pg/cell) (pg/cell) in Pellet (pg) PostWash_Pellet 1 0.3150.0000043 0.000083 1 × 10⁶ 83 PostWash_Pellet 2 6.941 0.000095PostWash_Pellet 3 11.008 0.00015

TABLE 8 Estimation of concentrations for compound example no. 5 in theCell Pellet compound compound Avg of compound Total compound example no.5 example no. 5 example no. 5 # of cells example no. 5 Sample (nM)(pg/cell) (pg/cell) in Pellet (pg) PostWash_Pellet 1 21.733 0.000610.00083 0.5 × 10⁶ 415 PostWash_Pellet 2 13.454 0.00038 PostWash_Pellet 346.529 0.0015

Example C1: Human Corneal Endothelial Cell Isolation

Research-consented cadaveric human corneas were obtained from eye banks.The corneal endothelium cell (CEC) layer and Descemet's membrane (DM)were scored with a surgical-grade reverse Sinsky endothelial stripper.The DM-endothelium cell layer was carefully peeled off the cornealstroma and cells were dissociated from the DM using 1 mg/ml collagenaseat 37° C. until cell detachment became apparent by microscopicobservation (45 minutes to 3 hours). Cells obtained in this manner wereused in the Examples C1-C17-below.

Example C2: Exposure of Cells to LATS Inhibitors and Measurement ofIntracellular YAP Distribution

Cells obtained as described in Example C1 were plated in glass-bottomblack wall 24-well dishes in corneal endothelial cell culture medium(human endothelial SF (serum free) medium (Invitrogen) with human serum)supplemented with LATS inhibitor compound example no. 133 or compoundexample no. 49 at a concentration of 10 micromolar or supplemented inDMSO as a negative control. Cells were cultured under these conditionsfor 24 hours at 37° C. in 5% CO2.

To measure the effect of the LATS inhibitors on the downstream targetYAP, intracellular YAP distribution was analyzed byimmunohistochemistry. Cell cultures were fixed with 4% PFA for 20minutes, permeabilized and blocked in a blocking solution of 0.3% TritonX-100 (Sigma-Aldrich) and 3% donkey serum in PBS for 30 minutes. Cellswere then labeled with primary antibody in the blocking solution for 12hours at 4° C. Primary antibody used was anti-YAP from Santa CruzBiotechnology. Samples were washed in PBS three times and donkey-raisedsecondary antibody Alexa Fluor 488 (Molecular Probes) at 1:500 dilutionwere applied for 30 minutes at room temperature. Negative control wasomitted primary antibody (data not shown). Fluorescence was observedusing a Zeiss LSM 880 confocal microscope.

YAP immunostaining is stronger in CECs exposed to cell proliferationmedium with the LATS inhibitor compound example no. 133 or example 49 asindicated by immunofluorescence staining of YAP, as shown in FIG. 14 .These compounds therefore had an effect on the intracellularlocalisation of the downstream target YAP.

Example C3: Exposure of Cells to LATS Inhibitors and Measurement of YAPPhosphorylation

Cells obtained as described in Example C1 were detached from the culturedish with 1 mg/ml collagenase for 15 minutes at 37° C., cell suspensionswere rinsed by centrifugation and plated in corneal endothelial cellculture medium (human endothelial SF medium (Invitrogen) with humanserum) in 6-well plates (Corning) and cultured for 2 to 4 days. Themedium was then replaced by fresh corneal endothelial cell culturemedium (human endothelial SF medium (Invitrogen) with human serum)supplemented with LATS inhibitor, compound example no. 133 or example no49 (diluted in DMSO) at a concentration of 10 micromolar or as anegative control DMSO alone without compound. Cells were cultured underthese conditions for 1 hour at 37° C. in 5% CO2.

Cell pellets were obtained by trypsin dissociation and centrifugationand washed with PBS. The pellets were lysed with 30 micromolar of RIPAlysis buffer containing protease inhibitor cocktail (Life Technologies)for 30 minutes, with vortexing every 10 minutes. The cell debris werethen pelleted at 4° C. for 15 minutes at 14 k rpm and the protein lysatewas collected. Protein concentration was quantified using a micro BCAkit (Pierce). Fifteen (15) micrograms of total protein was loaded ineach well of 4-20% TGX gels (BioRad) and Western blotting was performedaccording to the manufacturer's instructions. Membranes were probed withphospho-YAP (ser127) (CST, 1:500) or total Yap (Abnova, 1:500) antibodyand actin (Abcam) labelling was used as loading control. Membranes werestained with HRP-conjugated secondary antibodies, rinsed and imagedusing a ChemiDoc system (Biorad) according to the manufacturer'sinstructions.

Western blot analysis showed that both compound example no. 133 andexample no 49 caused a reduction in YAP phosphorylation levels in humanCECs. A marked difference was observed after one hour of treatment withcompound example no. 133 and example no 49 in human CECs, as shown byWestern Blot in FIG. 15 a . FIG. 15 b shows by graphical representationthe phosphorylated YAP levels normalized to beta-actin and FIG. 15 cshows phosphorylated YAP levels normalized to total YAP.

These results suggest that the LATS inhibitors compound example no 133and example no 49 can activate YAP signaling in human CECs.

Example C4: Human Corneal Endothelial Cell Population Expansion andMeasurement of Cell Density

Cells obtained as described in Example C1 were detached from the culturedish with 100 microlitres of Accutase (ThermoFisher) for 10 minutes at37° C., cell suspensions were rinsed by centrifugation and plated incorneal endothelial cell culture medium (human endothelial SF medium(Invitrogen) with human serum) in 6-well plates (Corning) supplementedwith LATS inhibitor compound example no. 133 or example no 49 (dilutedin DMSO) at a concentration of 10 micromolar or as a negative controlDMSO alone without compound. Cells were cultured at 37° C. in 5% CO2.

To measure cell proliferation, an Incucyte machine was used followingthe manufacturer's instructions (Essen Biosciences) to perform real-timequantitative live-cell analysis of cell confluence every 3 hours for 10days.

FIG. 16 shows the percentage confluence of the cell population over timeafter exposure to LATS inhibitor or DMSO alone. Although human CECs arenormally non-proliferative, the results show that both LATS inhibitorscompound example no. 133 and example no 49 could activate proliferationin these cells.

Example C5: Human Corneal Endothelial Cell Population Expansion andMeasurement of Cell Density

Cells obtained as described in Example C1 were plated in 48-well plates(Corning) in X-VIVO15 medium supplemented with LATS inhibitors as listedin Tables 9 and 10 (diluted in DMSO) at a concentration of 10 micromolaror as a negative control DMSO alone without compound. Cells werecultured at 37° C. in 5% CO2.

For each compound, two sets of cultures were generated. A first set ofcultures was fixed in 4% PFA for 20 minutes at room temperature aftercells isolated from the cornea had attached to the cell culture dish(typically 24 h after cell plating). A second set of cultures was fixedin 4% PFA for 20 minutes at room temperature 10 days after the firstone.

To measure cell density in all fixed cultures, the number of nucleistained with Sytox Orange (ThermoFisher) were counted per surface areaas follows: fixed cell cultures fixed were permeabilized in a solutionof 0.3% Triton X-100 (Sigma-Aldrich). Cells were then labeled in asolution of 0.5 micromolar of Sytox Orange in PBS for 5 minutes at roomtemperature. Nuclei were counted under a Zeiss epifluorescencemicroscope. The expansion factor was then determined by calculating theratio of the expanded population of cells to population of seeded cells.

The cell population expansion achieved with the tested compounds isshown by Tables 9 and 10 below.

TABLE 9 Fold Cell expansion Compound example no. Expansion factor Ex. 12521 Ex. 261 461 Ex. 47 449 Ex. 48a 446 Ex. 49 426 Ex. 5 408 Ex. 62 402Ex. 6 391 Ex. 14 337 Ex. 288 302 Ex. 66 280 Ex. 133 273 Ex. 287 237 Ex.290 221 Ex. 65 203 Ex. 17 187 Ex. 139 107 Ex. 289 84 Ex. 11 79 Ex. 48b21 Ex. 33 12 DMSO 7

TABLE 10 Endothelial Cell density in vitro (cells/mm² area) Compoundexample Cell density: number cells/mm² Ex. 12 4226 Ex. 261 4308 Ex. 474294 Ex. 48a 4021 Ex. 49 3873 Ex. 5 3911 Ex. 62 3301 Ex. 6 3378 Ex. 143271 Ex. 288 2779 Ex. 66 2503 Ex. 133 2028 Ex. 287 1893 Ex. 290 2071 Ex.65 1702 Ex. 17 1628 Ex. 139 1179 Ex. 289 1421 Ex. 11 1121 Ex. 48b 869Ex. 33 25 DMSO 13

Example C6: siRNA Knockdown of LATS1 and LATS2 in Human CornealEndothelial Cells

Cells obtained as described in Example C1 were plated in 24-well plates(Corning) in X-VIVO15 medium (Lonza). Cells were cultured at 37° C. in5% CO2. LATS1 and LATS2 were knocked down by transfection (lipofection,using RNAiMax, Thermofisher). Each well of the cell culture plate wastransfected with 0.5 micrograms of pools of 4 siRNAs targeting eachgene. siRNAs used in this study were Qiagen's LATS1 siRNA S100067172 andLATS2 siRNA S100106925. Scrambled siRNAs were used as negative controlsaccording to the manufacturer's protocol (Qiagen).

In order to measure cell proliferation, EdU staining was performed 48hours after transfection with LATS1 and LATS2 siRNAs or scrambled siRNAcontrols according to the manufacturer's instructions (LifeTechnologies). Cell nuclei were labeled with Sytox Orange. EdU and SytoxOrange fluorescence was observed using a Zeiss LSM 880 confocalmicroscope in order to measure the percentage of EdU-positive cellnuclei.

FIG. 17 shows that the percentage of EdU-positive CEC increased uponLATS1 and LATS2 knockdown, showing that knockdown of LATS leads to CECproliferation.

Example C7: Human Corneal Endothelial Cell Population Expansion andImmunohistochemical Observation of Cellular Morphology

Cells obtained as described in Example C1 were plated in 24-well plates(Corning) in corneal endothelial cell culture medium (human endothelialSF medium (Invitrogen) with human serum) supplemented with LATSinhibitor compound example no 49 (diluted in DMSO) at a concentration of10 micromolar or supplemented with a negative control of DMSO withoutcompound. Cells were cultured at 37° C. in 5% CO2 for 10 days.

In order to observe that the expanded cell population has the requiredcellular morphology for use in vivo, the ability of the cells to formtight junctions was measured by immunohistochemistry as follows. Cellcultures were fixed with 4% PFA for 20 minutes, permeabilized andblocked in a blocking solution of 0.3% Triton X-100 (Sigma-Aldrich) and3% donkey serum in PBS for 30 minutes. Cells were then labeled withprimary antibody in the blocking solution for 12 hours at 4° C. Primaryantibody used was ZO-1 from Invitrogen. Samples were washed in PBS threetimes and donkey-raised secondary antibody Alexa Fluor 488 (MolecularProbes) at 1:500 dilution were applied for 30 minutes at roomtemperature. Cells were washed three times in PBS and nuclei (DNA) werestained with Sytox Orange (567 nm, Life Technologies). Negative controlwas omitted primary antibody (data not shown). Fluorescence was observedusing a Zeiss LSM 880 confocal microscope.

CECs proliferated in the presence of medium and DMSO control show signsof polymegatism characteristic of dysfunctional CECs (FIG. 18A). CECsexposed to cell proliferation medium with the LATS inhibitor example no49 retained a normal corneal endothelial cell morphology and the abilityto form tight junctions as indicated by immunofluorescence staining oftight-junction marker Zonula Occludens-1 (ZO-1), as shown in FIG. 18B.Both a normal corneal endothelial cell morphology and the ability toform tight junctions are crucial for the maintenance of cornealendothelium functions.

Example C8: Human Corneal Endothelial Cell Population Expansion andMeasurement of Markers Collagen 8a2, AQP1, SLC4A11. RPE65, CD31, andNa/K ATPase

In order to verify that the expanded cell population expresses genesnormally expressed by corneal endothelial cells in vivo, cells werecultured and RT-PCR analysis was then performed to measure theexpression levels of Collagen 8a2, AQP1, SLC4A11, RPE65 and CD31.Immunohistochemical analysis was also performed to analyse the levels ofNa/K ATPase and Collagen 8a2 as follows.

Cells obtained as described in Example C1 were plated in 24-well plates(Corning) in corneal endothelial cell culture medium (human endothelialSF medium (Invitrogen) with human serum) supplemented with LATSinhibitor compound example no 49 (diluted in DMSO) at a concentration of10 micromolar or supplemented with a negative control of DMSO withoutcompound. Cells were cultured at 37° C. in 5% CO2 for 10 days. For thenegative control, dermal fibroblasts (Lonza) were cultured in DMEM-F12(LifeTechnologies) without LATS inhibitors.

To perform the RT-PCR analysis, total RNA was extracted using Trizol(Invitrogen), RNeasy Mini and QIA Shredder (Qiagen) according tomanufacturer's protocol. RNA quality and quantity were measured usingNanodrop 100 (Thermo-Fisher Scientific) and Bioanalyzer 2100 (AgilentTechnologies). cDNA was prepared by reverse transcription and relativemRNA expression was assessed by quantitative RT-PCR using an 7900HTsystem (Applied Biosystems). The following cycling parameters were used:

-   -   1) 50° C. for 2 minutes; 2) 95° C. for 10 minutes; 3) 95° C. for        15 seconds 4) 60° C. for 1 minute.

Steps 3-4 were repeated 40 times.

Actin mRNA levels were measured and used as a endogenous controls tonormalize gene expression levels and calculate delta Ct values accordingto the formula dCt=Ct for gene of interest—Ct for endogenous control.Primers were obtained from Applied Biosystems.

RT-PCR analysis as shown in FIG. 19 indicates that a corneal endothelialcell population expanded with LATS inhibitor compound example no 49express genes normally expressed by corneal endothelial cells in vivo,including Collagen 8a2, AQP1, SLC4A11. The cells do not express markersof other epithelia present in the eye, including RPE65 (a marker ofretinal pigmented epithelium) and CD31 (a marker of vascularepithelium).

To check the expression of Na/K ATPase and Collagen 8a2 byimmunohistochemistry the cell cultures were fixed with 4% PFA for 20minutes, permeabilized and blocked in a blocking solution of 0.3% TritonX-100 (Sigma-Aldrich) and 3% donkey serum in PBS for 30 minutes. Cellswere then labeled with primary antibodies in the blocking solution for12 hours at 4° C. Primary antibodies used were Na/K ATPase and Collagen8a2 (Santa Cruz Biotechnology). Samples were washed in PBS three timesand donkey-raised secondary antibody Alexa Fluor 488 or 647 (MolecularProbes) at 1:500 dilution were applied for 30 minutes at roomtemperature. Cells were washed three times in PBS and nuclei (DNA) werestained with Sytox Orange (567 nm, Life Technologies). Negative controlswere either omitted primary antibody or isotype control antibody (datanot shown). Fluorescence was observed using a Zeiss LSM 880 confocalmicroscope.

Immunohistochemical analysis as shown in FIG. 20 indicates that acorneal endothelial cell population expanded with LATS inhibitorcompound example no 49 express genes normally expressed by cornealendothelial cells in vivo, including Na/K ATPase (FIG. 20 a ) andCollagen 8a2 (FIG. 20 b ).

Example C9: Human Corneal Endothelial Cell Population Expansion andMeasurement of Markers CD44, CD105, CD166 and CD73

In order to verify that the expanded cell population does not undergoendothelial to mesenchymal transition, CECs were cultured as follows andFACS analysis was then performed to analyse the levels of CD44, CD105,CD166 and CD73.

To generate mature CEC cultures, cells obtained as described in ExampleC1 were plated in X-VIVO15 supplemented with LATS inhibitor compoundexample no. 48a (diluted in DMSO) at a concentration of 3 micromolar.Cells were cultured in 48 well plates (corning) at 37° C. in 5% CO2 fortwo weeks. Cells were passaged twice during this time by detaching cellswith 1 mg/ml collagenase for 15 minutes at 37° C., rinsing cellsuspensions by centrifugation and plating in fresh medium. These cellswere then grown in XVIVO medium without a LATS inhibitor for a further 2weeks.

To measure the levels of CD44, CD105, CD166 and CD73 by FACS the CECswere treated with Accutase (ThermoFisher, Cat #A1 110501) for 20 minutesin 5% CO2 at 37° C. The reaction was stopped by using cell culturemedium containing 10% Serum and transferred to a falcon tube for acentrifugation step (1000 rpm, 5 minutes). After aspirating the mediumcells were resuspended in 200 microlitre FACS buffer (PBS/10% FBS).

Antibodies against CD44, CD105, CD166 and CD73 (BD Biosciences) wereadded to the cell suspension and incubated for 30 minutes on ice. Cellswere washed 3 times after antibody labelling with FACS buffer andresuspended in 500 microlitre in FACS buffer. Before FACS sorting, cellswere filtered through a 70 micrometre filter and stored on ice untilsorting. Cells were sorted on a BD FACSAria II instrument. FACS datawere analyzed using BD FACSDiva software.

FACS analysis as shown in FIG. 21 indicates that the FACS markerexpression profile of the cell population expanded in the presence ofthe LATS inhibitor is different from the FACS marker expression profileof cells that have undergone endothelial to mesenchymal transition.Specifically, cells cultured in the presence of the LATS inhibitorexpress lower levels of CD44, CD73, CD105 and CD166 compared to cellsgrown in the absence of the LATS inhibitor. Importantly, CD73 is amarker of endothelial to mesenchymal transition, therefore these resultsindicate that CECs cultured in the presence of LATS inhibitors do notundergo endothelial to mesenchymal transition.

Example C10: Cell Bioprinting: GelMA Preparation Synthesis of GelMA

Gelatin methacrylate (GelMA) with approximately 100% of Lys residuesmethacrylated was synthesized according to a previously publishedprotocol (Nichol, J. W. et al. Biomaterials, 2010, p. 5536-5544). 20grams of porcine derived gelatin (Cat #G2500, Sigma) was dissolvedovernight at 50° C. in 200 ml of PBS without calcium and magnesium(DPBS, Cat #21-031, Corning). With strong agitation, methacrylicanhydride (Cat #276685, Sigma) was added dropwise (approximately 1ml/min) into the gelatin solution to reach the concentration of 8%(vol/vol). The mixture was stirred at 60° C. in an oil bath for 3 hoursbefore adding 200 ml of the DPBS and followed by thorough mixing for anadditional 15 minutes. The diluted mixture was purified via dialysisagainst Milli-Q water (used 15 kDa MWCO Spectra/Por dialysis tubing) for1 week at 45° C. to remove methacrylic acid. The purified samples werelyophilized and stored at −80° C. until further use.

¹H NMR (400 MHz, D₂O, 35° C.) was used to determine the degree ofmethacrylation for a 15 mg/mL solution of GelMA. The peak area ratio ofPhe to methacrylamide was found to be 1.00:0.82 by comparing the area ofthe multiplet at 7.25-7.50 ppm (assumed to be Phe protons, 5H per Phe inGelMA) to the sum of the areas of four singlets at 5.47 ppm, 5.51 ppm,5.71 ppm, and 5.76 ppm (assumed to be vinyl protons, 2H permethacrylamide in GelMA). The molar Phe:Lys ratio in the gelatin used inthis reaction was determined to be 1.00:2.06 by amino acid analysis.Therefore, the molar ratio of Phe:Lys:methacrylamide in GelMA is1.00:2.06:2.05. Assuming that the primary site of gelatin methacrylationis at Lys residues, the 1H NMR analysis indicates the degree ofmethacrylation is ˜100% (2.05/2.06×100%=99.5%).

Stock solutions of GelMA and LAP were prepared in DPBS, pH-adjusted,sterile filtered, and stored at 4° C. until further use as describedbelow. The following protocol exemplifies the preparation of a 15% w/vGelMA and 0.15% w/v LAP stock, but other strengths were preparedfollowing an identical procedure. To prepare a 15% w/v GelMA stocksolution, 1.5 gram of the freeze-dried GelMA prepared as described abovewas dissolved in 10 ml of pre-warmed DPBS at 37° C. After the GelMA wasfully dissolved, the photoinitiator was introduced by adding into theGelMA solution 15 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP), resulting in a stock solution containing 15% w/v GelMA and 0.15%w/v LAP. LAP was synthesized using published procedure (Biomaterials2009, 30, 6702-6707). 500 microlitres of 1N NaOH (Cat #BDH-7222-1, VWR)was added to the solution to adjust the pH to neutral before thesolution was filtered using 0.22 micrometre sterile membranes(Millipore). The final filtrate was separated into 500 ul aliquots andstored at 4° C. until further use.

Example C11: Bioprinters: Design and Operation

In order to develop a cell therapy where the location of cell deliveryis precisely controlled, a bioprinting technology is used to preciselyposition CECs on the posterior side of the cornea.

Bioprinting experiments were performed on glass coverslips using HEK293cells. Red-fluorescent protein-labeled HEK293 cells were cultured toconfluence in 6-well culture plates in DMEM-F12 with 5% fetal bovineserum. Cells were then detached from the culture dish with 100microlitre of TripLE (Invitrogen) for 10 minutes at 37° C., cellsuspensions were rinsed by centrifugation and resuspended in DMEM-F12 ata concentration of 80 million cells per ml.

A first bioprinter was designed based on dynamic light projection (DLP)technology and the schematic of the system is illustrated in FIG. 26 .The system consists of five main components: 1. A UVA light source (365nm, S2000, EXPO); 2. A digital micromirror array device (DMD, DLP-07XGA; Texas Instruments) to modulate light to generate light patterns; 3.An optical system to project the light pattern towards the sample; 4. Anautomated stage to move in all three axes in a synchronized manner withthe corresponding light patterns generated by the DMD; 5. A computerwhich feeds the DMD chip image flows and controls all the components.

For bioprinting, a HEK293 cell/biomatrix mixture was prepared by mixing50 microlitre of the 15% GelMA stock solution containing 0.15% LAP withthe same volume of HEK293 cell suspension to reach a final cell densityof 40 million per ml. The cell/GelMA mixture was then added to thecenter of a glass coverslip and polymerized in the shape of the letter“e” by applying 365 nm UVA light mask for 30 seconds. Images were thenacquired using a Zeiss LSM 880 confocal microscope.

Results showed that cell-laden constructs could be precisely bioprintedin the form of the letter “e” (actual results are shown on the bottomleft hand side of FIG. 26 : the “e”). This indicated that thebioprinting technology could enable precise control of the location ofcell delivery.

In order to facilitate in vivo animal work, the core technology of thebioprinter described above was converted into a compact portablehandheld design as follows. A light emitting head was composed of a highpower LED emitter (365 nm, LED Engin) and an aspherical lens to providecollimated light beam. The light emitting head was built in a customizedaluminum case for better heat dissipation. A rechargeable battery, powerswitch and other electronics were accommodated in a 3D printed plastichandle, which was assembled together with the light emitting head via apivot to enable more freedom to turn the head. 3D printed adaptorsallowed attaching to the head light guides of different dimensions tocontrol the size of the illumination area. Inspired by the DMD basedsystem, this handheld device can use masks printed on transparency sheetattached to the front of the head to generate customized light patternto control photopolymerization.

Example C12: Bioprinting Cell Laden Constructs on the Posterior Side ofthe Human Cornea Ex Vivo

In order to determine whether the portable handheld bioprinting devicewould enable the precise positioning of cells on the posterior side ofthe human cornea, bioprinting experiments were preformed ex vivo usingHEK293 cells and human corneas obtained from eye banks. Red-fluorescentprotein-labeled HEK293 cells were cultured to confluence in 6-wellculture plates in DMEM-F12 with 5% fetal bovine serum. Cells were thendetached from the culture dish with 100 microlitres of TripLE(Invitrogen) for 10 minutes at 37° C., cell suspensions were rinsed bycentrifugation and resuspended in DMEM-F12 at a concentration of 80million cells per ml.

For bioprinting, a HEK293 cell/biomatrix mixture was prepared by mixing50 microlitre of the 15% GelMA stock solution containing 0.15% LAP withthe same volume of HEK293 cell suspension to reach a final cell densityof 40 million per ml.

The cell/GelMA mixture was then added to the center of a glass coverslipand polymerized in the shape of the letter “e” by applying 365 nm UVAlight mask for 30 seconds. Images were then acquired using a Zeiss LSM880 confocal microscope.

Human donor corneas obtained from eye banks were removed from theirstorage solution (Optisol) and rinsed briefly with DPBS. Under thedissecting microscope, the Descement membrane was carefully scrapedbefore the corneas were further rinsed with DPBS. One cornea was thenplaced on a plastic lid of a 35 mm petri dish with the inner surfacefacing up. A cell/biomatrix mixture was prepared freshly by mixing 50microlitres of the 15% GelMA stock solution containing 0.15% LAP withthe same volume of HEK-293-RFP cell suspension to reach the final celldensity of 40 million per ml. The cell/GelMA mixture was then added tothe center of the cornea and then a convex shaped plastic holder wasapplied to form a closed chamber mimicking the eye's anterior chamber.The whole construct was then carefully flipped and the handheldbioprinting device was used to project a 5.5-mm diameter circularpattern of 365 nm UVA light through the cornea for 30 seconds. Thispattern of UV light was expected to polymerize the cell/GelMA mixture inthe form of a disk attached to the posterior side of the cornea. Inorder to determine whether this occurred, we separated the cornea fromthe lid, rinsed any unpolymerized and unattached material by pipettingDPBS on the posterior side of the cornea and images were acquired usinga Zeiss LSM 880 confocal microscope.

Results show that after unpolymerized and unattached material wasrinsed, a circular pattern of red-fluorescent protein-labelled cells wasretained on the posterior side of the cornea (FIG. 27 ). This confirmedthat cells could be bioprinted on the posterior side of the cornea byusing a handheld device that projects 365 nm UVA light through thecornea.

Example C13: Bioprinting Cell Laden Constructs on the Posterior Side ofthe Rabbit Cornea In Vivo

CEC preparation

Cells obtained as described in Example C1 were detached from the culturedish with 100 microlitres of Accutase (ThermoFisher) for 10 minutes at37° C., cell suspensions were rinsed by centrifugation and plated inX-VIVO15 supplemented with LATS inhibitor compound example no. 48a(diluted in DMSO) at a concentration of 3 micromolar. Cells werecultured in 6-well plates (corning) at 37° C. in 5% CO2 for two weeks.Cells were passaged twice during this time by detaching cells with 1mg/ml collagenase for 15 minutes at 37° C., rinsing cell suspensions bycentrifugation and plating in fresh medium. These cells were then grownin XVIVO medium without a LATS inhibitor for a further 2 weeks to createmature CECs.

In order to prepare CECs for bioprinting inside the rabbit eye, acell/biomatrix mixture was generated by mixing 15 microlitres of the 15%GelMA stock solution containing 0.15% LAP with 30 microlitres of CECsuspension to reach the final cell density from 0.625 million per ml to25 million per ml. The cell/GelMA mixture was then added to the centerof the cornea.

Rabbit Model of Corneal Endothelial Cell Deficiency

Corneal endothelial cell deficiency was unilaterally created in theright eye of NZA rabbits by scraping off the whole cornea endotheliumusing a silicon tip needle. The anterior chamber was rinsed using anaspiration cannula to remove floating debris. Forty microliters offreshly prepared cell/GelMA mixture was injected into the anteriorchamber and followed by 365 nm UVA exposure for 30 seconds using thehandheld bioprinter equipped with a 3 mm light guide about 1 cm awayfrom the ocular surface. Gentle rinsing was performed using a smallcannula to inject 500 microlitres of heparin supplemented balanced saltsolution to remove unpolymerized material and floating cells. The lefteye of each rabbit was left intact and served as a control. After celldelivery, rabbits received analgesic treatment (Tramadol 10 mg/kg PO BIDin first 2 weeks, Meloxicam 0.3 mg/kg PO SID in first 2 weeks or as longas the animal showed signs of ocular discomfort), anti-inflammatorytreatment (Ancef® (cefazolin) 50 mg sub-Tenon immediate post-procedure,Tobrex® ophthalmic solution t.i.d in 1st week and b.i.d thereafter,ampicillin 80 mg/kg/day (40 mg BID) SQ for first week) andimmunosuppression (Cyclosporine A (0.5%) top oc. t.i.d 1st week andb.i.d. thereafter, Gentocin®-Durafilm® (Gentamicin sulfate andbetamethasone, MERCK) top oc. t.i.d 1st week and b.i.d. thereafter,Cyclosporine A (5 mg/kg/day) SQ in 1st week and then % dosagethereafter).

Rabbits were sacrificed three weeks after cell delivery, corneas weredissected and fixed in 4% PFA. Immunohistochemistry was performed todetect the presence of human nuclear antigen (Millipore) to confirm thepresence of human cells and ZO-1 (Invitrogen) to determine whetherbioprinted CECs could form tight junctions observed in a normal cornealendothelioum. Images were then acquired using a Zeiss LSM 880 confocalmicroscope.

Results indicated that in experimental rabbits, the corneal endotheliumstructure can be detected using ZO-1 immunohistochemistry (FIG. 28 ,panel A). In the right eye of a rabbit where the corneal endothelium wassurgically removed and no CEC was bioprinted, the ZO-1 staining isabsent, indicating an absence of normal corneal endothelium structure(FIG. 28 , panel B). In the right eye of a rabbit where the cornealendothelium was surgically removed and CEC were bioprinted, the ZO-1staining is present, indicating that a corneal endothelium structure hasbeen rebuilt (FIG. 28 , panel C).

Human nuclear antigen staining was used to confirm that the cells thatrebuilt the corneal endothelium in FIG. 28 , panel C are the human CECsdelivered by bioprinting. FIG. 28 , panels D and E show that humannuclear antigen immunostaining is absent in eyes that did not receiveany human CECs. In contrast, human nuclear antigen-positive cells coverthe imaged field in eyes where human CECs were bioprinted (FIG. 28 ,panel F), indicating that the ZO-1-labeled corneal endothelium structureshown in FIG. 28 , panel C is composed of the human CECs bioprinted onthe posterior side of the rabbit cornea.

Example C14: Bioprinting Cell Laden Constructs of Highly CustomizableShapes on the Posterior Side of the Human Cornea Ex Vivo

Human donor corneas obtained from eye banks were removed from theirstorage solution (Optisol) and rinsed briefly with DPBS. Under thedissecting microscope, the Descement membrane was carefully scrapedbefore the corneas were further rinsed with DPBS. One cornea was thenplaced on a plastic lid of a 35 mm petri dish with the inner surfacefacing up.

A cell/biomatrix mixture was prepared freshly by mixing 50 μl of the 15%GelMA stock solution containing 0.15% LAP with the same volume ofHEK-293-RFP cell suspension to reach the final cell density of 40million per ml. The cell/GelMA mixture was then added to the center ofthe cornea and then a convex shaped plastic holder was applied to form aclosed chamber mimicking the eye's anterior chamber. The whole constructwas then carefully flipped and the DLP bioprinting device mentioned inExample C11 was used to project customizable patterns of 365 nm UVAlight through the cornea for 7.5 seconds. Any unpolymerized andunattached material was then rinsed by DPBS on the posterior side of thecornea and images were acquired using a Zeiss fluorescencestereomicroscope.

In order to demonstrate the reproducibility and customizability of ourbioprinting technology, light patterns in the design of differentletters, which can be easily recognized, were projected to the samples.Results (FIG. 29 ) show that customizable cell laden constructs withhigh precision can be bioprinted to the posterior side of the corneadetermined by the light patterns through the cornea.

Example C15: Residual Compound Estimation Study Standard CurvePreparation

A dilution series of stock standards were made at concentrations of 30micromolar, 3 micromolar, 300 nM, 30 nM, 3 nM, 0.3 nM and 0 micromolar.Compound example 48a (10 mM in DMSO) stock was spiked into 50%acetonitrile 50% water to make the spike standards. The spikingstandards were used to spike the blank media samples. 10 microlitre ofspiking standard was added to 90 microlitres of media to create thespiked media standards. 3000 nM, 300 nM, 30 nM, 3 nM, 300 μM, 30 μM, and0 nM media standards were used to generate the compound example no. 48astandard curve.

50 microlitre of each media sample was treated with 300 microlitre ofthe extraction solution. The extraction solution consists ofacetonitrile/methanol (3:1). The standard curve samples were extractedusing the same volumes and conditions as the unknown media samples. Theextracted samples were centrifuged at 10,000 rpm for 5 min. Aftercentrifugation, 200 microlitres of each sample supernatant wastransferred to a clean 96-well plate. The extracted samples wereanalyzed by High Resolution LC-MS. Thermo Xcalibur Software and QuanBrowser were used to generate the standard curve and calculate theconcentration values. An external calibration method using linearlog-log scaling was used to calculate the concentration of compoundexample no. 48a in the culture media samples.

LC-MS Analysis

High-resolution chromatography was performed using the Thermo UltimateUPLC and a Kinetex 2.1×50 mM C18 RP column (2.6 micron particles). Themobile phases consisted of 5 mM Ammonium Acetate in H₂O for Buffer A,and 0.1% Formic Acid in Acetonitrile for Buffer B. A standard binarylinear gradient was used for Reverse Phase Chromatography. The Thermo QExactive mass spectrometer was used for this study. The UPLC effluentwas directed into the Q Exactive Mass Spectrometer which was equippedwith an ESI ion source. The mass spectrometer was programmed to performin High Resolution Full Scan mode. Extracted Ion Chromatograms were usedfor chromatographic integration.

Test System

Cell preparation for the CEC washout: the washout experiment wasperformed in triplicates, where each sample contained 0.5 million cells.All samples (media only, media with cells, supplemented with compoundexample no. 48a and cells in media supplemented with compound exampleno. 48a) were cultured at 37 degree incubator for the duration of theexperiment. The medium used for the experiment was X-VIVO 15 (Lonza, catno. 04-744Q).

The cells were cultured to passage 2 in X-VIVO-15 medium supplementedwith 3 micromolar of compound example no. 48a. Upon reaching confluency,the medium was removed by aspiration and replaced with fresh medium notsupplemented with compound example no. 48a. The cells were cultured foran additional week in the absence of compound example no. 48a. After oneweek of culture in the absence of the compound, the medium was removedby aspiration, refreshed with the medium without the compound and cellculture was continued in the absence of the compound for another sixdays. On day 7, the following samples were generated:

-   -   5. 2 ml of “blank” medium (not supplemented with the compound)    -   6. 2 ml of medium cultured in the presence of the compound        (pre-wash)    -   7. 2 ml of media for each wash sample 1-10    -   8. Cell pellets (post-wash)

The cells were collected by using the cell lifter (Costar, cat #3008).

The media and cell pellet samples were analyzed and quantified usingHigh Resolution LC-MS.

To estimate the residual levels of compound example no. 48a, cellpellets and supernatant were collected. The amount of compound exampleno. 48a was determined by LC-MS.

Estimation of compound example no. 48a concentrations in the Pre-washMedia, Wash Media (Table 11) and the Cell Pellet are summarized (Table12). Pre-wash Media samples have the highest levels of compound exampleno. 48a (approx. 35 nM to 122 nM range). Post-wash Pellet levels havelow, but detectable levels of compound example no 48a (picomolar).

TABLE 11 Estimation of concentrations for compound example no. 48a inthe Pre-wash Media and Wash Media compound compound compound example no.example no. example no. 48a (nM) 48a (nM) 48a (nM) Volume in Triplicatein Triplicate in Triplicate Sample (mL) #1 #2 #3 Pre-wash Media 2 35.34466.248 121.863 Wash Media 1 2 0.408 0.614 0.712 Wash Media 2 2 BLQ 0.0030.002 Wash Media 3 2 BLQ BLQ BLQ Wash Media 4 2 BLQ BLQ BLQ Wash Media 52 BLQ BLQ BLQ Wash Media 6 2 BLQ BLQ BLQ Wash Media 7 2 BLQ BLQ BLQ WashMedia 8 2 BLQ BLQ BLQ Wash Media 9 2 BLQ BLQ BLQ Wash Media 10 2 BLQ BLQBLQ

TABLE 12 Estimation of concentrations for compound example no. 48a inthe Cell Pellet compound compound Avg of compound Total of compoundexample no. example no. example no. 48a # of cells example no. 48aSample 48a (nM) 48a (pg/cell) (pg/cell) in Pellet (pg) PostWash_Pellet 10.004 0.13 × 10⁻⁶  1.5 × 10⁻⁶ 0.5 × 10⁶ 0.75 PostWash_Pellet 2 0.071 2.3× 10⁻⁶ PostWash_Pellet 3 0.065 2.1 × 10⁻⁶

The amount of compound example no. 48a in the Pre-wash Media from threeincubations ranges from 35.344-121.863 nM. The average amount ofcompound example no. 48a in the Cell Pellet was 0.75 picograms for500,000 cells.

Example C16: Reducing Immune Rejection by AAV-Mediated Deletion of theBeta-2-Microglobulin Gene in LSCs AAV Vector Design:

The vector map shown in FIG. 30 provides the design of the AAV vectorused to express the CRISPR system in LSCs. The vector expressedStaphylococcus aureus Cas9 (Ran et al, Nature. 2015 Apr. 9;520(7546):186-191) and a B2M-specific guide RNA inserted in the Aar Irestriction site.

Cell transduction: LSCs obtained as described in Example 1 were culturedin X-VIVO15 medium supplemented with the LATS inhibitor compound exampleno 48a. LSC were transduced either in suspension immediately afterisolation from human corneas or after isolation, attachment andexpansion in the LATS inhibitor compound to a confluency of 50-60% in a35 mm petri dish eight days after LSC isolation. LSCs were transduced atMOls of 10K, 50K, 100K or 200K. The vector particles were diluted in abuffer composed of 1×PBS+0.001% Pluronic and pipetted into the culturedish. The viral particle storage buffer alone represented thetransduction negative control. Cells were cultured for 72 hours in a 5%CO₂ incubator before FACS analysis and sorting was performed.

Facs Analysis:

LSCs were treated with Accutase (ThermoFisher, Cat #A1 110501) for 20minutes in 5% CO₂ at 37° C. After scraping the cells, the reaction 5 wasstopped by using cell culture medium containing 10% serum andtransferred to a falcon tube for a centrifugation step (1000 rpm, 5minutes). After aspirating the medium, cells were resuspended in 200microlitre FACS buffer (PBS/10% FBS).

To analyze the expression of B2M and HLA-ABC, 5 microlitre APC mouseanti-human Beta2-microglobulin antibody (Biolegend, Cat #316312) and 20microlitre PE mouse antihuman HLA-ABC antibody (BD Bioscience, Cat#560168) were added to the cell suspension and incubated for 30 minuteson ice. Cells were washed 3 times after antibody labelling with FACSbuffer and resuspended in 500 microlitres in FACS buffer.

Before FACS sorting, cells were filtered through a 70 micrometre filterand stored on ice until sorting. In order to prevent cells sticking tothe wall, collection tubes were filled with the serum for 30 minutesbefore the sort. Cells were sorted on a BD FACSAria II instrument into20 prepared collection tubes, using human serum enriched LSC medium.FACS data were analyzed using BD FACSDiva software.

Results presented in FIG. 31 show at an MOI of 200K, AAV-mediatedexpression of the CRISPR system enabled deletion of B2M and subsequentelimination of HLA A, B and C occurred in 24.9 percent of the LSCs whenattached cells were transfected and 20.0 percent of LSCs when cells insuspension were transduced.

Example C17: Reducing Immune Rejection by AAV-Mediated Deletion of theBeta-2-Microglobulin Gene in CECs AAV Vector Design:

The vector map shown in FIG. 30 provides the design of the AAV vectorused to express the CRISPR system in CECs. The vector expressedStaphylococcus aureus Cas9 (Ran et al, Nature. 2015 Apr. 9;520(7546):186-191) and a B2M-specific guide RNA inserted in the Aar Irestriction site.

Cell Transduction:

Cells obtained as described in Example C1 were detached from the culturedish with 100 microlitres of Accutase (ThermoFisher) for 10 minutes at37° C., cell suspensions were rinsed by centrifugation and plated incorneal endothelial cell culture medium (human endothelial SF medium(Invitrogen) with human serum) in 6-well plates (Corning) 10supplemented with LATS inhibitor compound example no. 133 or example no49 (diluted in DMSO) at a concentration of 10 micromolar or as anegative control DMSO alone without compound. Cells were cultured at 37°C. in 5% CO₂.

CECs were cultured in corneal endothelial cell culture medium (humanendothelial SF medium (Invitrogen) with human serum) in 6-well plates(Corning) supplemented with LATS inhibitor compound example no. 133 orexample no 49 (diluted in DMSO) at a concentration of 10 micromolar. CECwere transduced after isolation, attachment and expansion in the LATSinhibitor compound to a confluency of 50-60% in a 6-well plate eightdays after CEC isolation. CECs were transduced at MOls of 0.1K, 1K, 5K,10K, 20K or 40K. The vector particles were diluted in a buffer composedof 1×PBS+0.001% Pluronic and pipetted into the culture dish. The viralparticle storage buffer alone represented the transduction negativecontrol. Cells were cultured for 72 hours in a 5% CO₂ incubator beforeFACS analysis and sorting was performed.

Facs Analysis:

CECs were treated with Accutase (ThermoFisher, Cat #A1110501) for 20minutes in 5% CO₂ at 37° C. After scraping the cells, the reaction 5 wasstopped by using cell culture medium containing 10% serum andtransferred to a falcon tube for a centrifugation step (1000 rpm, 5minutes). After aspirating the medium cells were resuspended in 200microlitre FACS buffer (PBS/10% FBS).

To analyze the expression of B2M and HLA-ABC, 5 microlitre APC mouseanti-human Beta2-microglobulin antibody (Biolegend, Cat #316312) and 20microlitre PE mouse antihuman HLA-ABC antibody (BD Bioscience, Cat#560168) were added to the cell suspension and incubated for 30 minuteson ice. Cells were washed 3 times after antibody labelling with FACSbuffer and resuspended in 500 microlitres in FACS buffer.

Before FACS sorting, cells were filtered through a 70 micrometre filterand stored on ice until sorting. In order to prevent cells sticking tothe wall, collection tubes were filled with the serum for 30 minutesbefore the sort. Cells were sorted on a BD FACSAria II instrument into20 prepared collection tubes, using human serum enriched CEC culturemedium. FACS data were analyzed using BD FACSDiva software.

Results presented in FIG. 32 show that AAV-mediated expression of theCRISPR system enabled deletion of B2M and subsequent elimination of HLAA, B and C. At an MOI of 40K, B2M deletion occurred in 17 percent of theCECs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein. Unless indicated otherwise,each of the references cited herein is incorporated in its entirety byreference.

Claims to the invention are non-limiting and are provided below.Although particular embodiments and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims, or the scope of subject matter ofclaims of any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the disclosure without departing fromthe spirit and scope of the disclosure as defined by the claims. Thechoice of nucleic acid starting material, clone of interest, or librarytype is believed to be a matter of routine for a person of ordinaryskill in the art with knowledge of the embodiments described herein.Other embodiments, advantages, and modifications are considered to bewithin the scope of the following claims. Those skilled in the art willrecognize or be able to ascertain, using no more than routineexperimentation, many equivalents of the specific embodiments of theinvention described herein.

1-315. (canceled)
 316. A method comprising culturing a seedingpopulation of cells in the presence of a composition comprising a LATSinhibitor, wherein the seeding population of cells comprises ocularcells, and wherein culturing the seeding population of cells generatesan expanded population of cells.
 317. The method of claim 316, whereinthe LATS inhibitor is a compound of Formula A1, or a salt, orstereoisomer thereof,

wherein X¹ and X² are each independently CH or N; Ring A is (a) a 5- or6-membered monocyclic heteroaryl that is linked to the remainder of themolecule through a carbon ring member and comprises, as ring member, 1to 4 heteroatoms that are independently selected from N, O and S,provided that at least one of the heteroatom ring member is anunsubstituted nitrogen (—N═) positioned at the 3- or the 4-positionrelative to the linking carbon ring member of the 5-membered heteroarylor at the para ring position of the 6-membered heteroaryl; or (b) a9-membered fused bicyclic heteroaryl that is selected from

wherein “*” represents the point of attachment of ring A to theremainder of the molecule; wherein ring A is unsubstituted orsubstituted by 1 to 2 substituents independently selected from halogen,cyano, C₁₋₆alkyl, C₁₋₆haloalkyl, —NH₂, C₁₋₆alkylamino,di-(C₁₋₆alkyl)amino, C₃₋₆cycloalkyl, and phenylsulfonyl; R⁰ is hydroxylor C₁₋₆alkoxy; R¹ is hydrogen or C₁₋₆alkyl; R² is selected from (a)C₁₋₈alkyl that is unsubstituted or substituted by 1 to 3 substituentsindependently selected from (i) halogen; (ii) cyano; (iii) oxo; (iv)C₂alkenyl; (v) C₂alkynyl; (vi) C₁₋₆haloalkyl; (vii) —OR⁶, wherein R⁶ isselected from hydrogen, C₁₋₆alkyl that is unsubstituted or substitutedby R⁰ or —C(O)R⁰; (viii) —NR^(7a)R^(7b), wherein R^(7a) is hydrogen orC₁₋₆alkyl, and R^(7b) is selected from hydrogen, —C(O)R⁰, C₁₋₆alkyl thatis unsubstituted or substituted by —C(O)R⁰; (ix) —C(O)R⁸, wherein R⁸ isR⁰ or —NH—C₁₋₆alkyl-C(O)R⁰; (x) —S(O)₂C₁₋₆alkyl; (xi) monocyclicC₃₋₆cycloalkyl or polycyclic C₇₋₁₀cycloalkyl that are each unsubstitutedor substituted by 1 to 2 substituents independently selected fromhalogen, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆haloalkyl, R⁰, —NH₂,C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino; (xii) 6-memberedheterocycloalkyl comprising, as ring members, 1 to 2 heteroatomsindependently selected from N, O and S and that is unsubstituted orsubstituted by 1 to 2 substituents independently selected from hydroxyl,halogen, C₁₋₆alkyl, C₁₋₆alkylamino, and di-(C₁₋₆alkyl)amino; (xiii)phenyl that is unsubstituted or substituted by halogen; (xiv) 5- or6-membered monocyclic heteroaryl comprising, as ring members, 1 to 4heteroatoms independently selected from N and O; and (xv) 9- or10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2heteroatoms independently selected from N and O; (b) —S(O)₂C₁₋₆alkyl;(c) phenyl that is unsubstituted or substituted by 1 to 2 substituentsindependently selected from halogen, C₁₋₆alkyl and R⁰; (d)C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2substituents independently selected from C₁₋₆haloalkyl, R⁰,C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that isunsubstituted or substituted by R⁰ or —C(O)R⁰; and (e) 4-memberedheterocycloalkyl comprising, as ring members, 1 to 2 heteroatomsselected from N, O and S and that is unsubstituted or substituted by 1to 2 substituents independently selected from C₁₋₆haloalkyl, R⁰,C₁₋₆alkylamino, di-(C₁₋₆alkyl)amino, —C(O)R⁰, and C₁₋₆alkyl that isunsubstituted or substituted by R⁰ or —C(O)R⁰; or R¹ and R² can be takentogether with the nitrogen atom to which both are bound to form a 4- to6-membered heterocycloalkyl that can include, as ring members, 1 to 2additional heteroatoms independently selected from N, O, and S, whereinthe 4-to 6-membered heterocycloalkyl formed by R¹ and R² taken togetherwith the nitrogen atom to which both are bound is unsubstituted orsubstituted by 1 to 3 substituents independently selected from halogen,C₁₋₆alkyl, C₁₋₆haloalkyl, and R⁰; R³ is selected from hydrogen, halogenand C₁₋₆alkyl; and R⁵ is selected from hydrogen, halogen and —NH-(3- to8-membered heteroalkyl), wherein the 3- to 8-membered heteroC₃₋₈alkyl ofthe —NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms aschain members and is unsubstituted or substituted by R⁰.
 318. The methodof claim 317, wherein the LATS inhibitor is a compound of a formulaselected from Formulae I to IV:


319. The method of claim 318, wherein ring A is: (a) selected from thegroup consisting of

which are each unsubstituted or substituted by a substituent selectedfrom the group consisting of halogen, cyano, C₁₋₆alkyl, C₁₋₆haloalkyl,and —NH₂; or (b) is

 which is unsubstituted or substituted by C₁₋₆alkyl.
 320. The method ofclaim 318, wherein ring A is


321. The method of claim 318, wherein R² is selected from the groupconsisting of: (a) C₁₋₈alkyl that is unsubstituted or substituted by A1to A3 substituents independently selected from the group consisting of:(i) cyano; (ii) C₂alkynyl; (iii) C₁₋₆haloalkyl; (iv) —OR⁶, wherein R⁶ isselected from the group consisting of hydrogen, and C₁₋₆alkyl that isunsubstituted or substituted by R⁰ or —C(O)R⁰; (v) —NR^(7a)R^(7b),wherein R^(7a) is hydrogen or C₁₋₆alkyl, and R^(7b) is selected from thegroup consisting of hydrogen, —C(O)R⁰, and C₁₋₆alkyl that isunsubstituted or substituted by —C(O)R⁰; (vi) —C(O)R⁸, wherein R⁸ is R⁰;(vii) —S(O)₂C₁₋₄alkyl; (viii) monocyclic C₃₋₆cycloalkyl that isunsubstituted or substituted by a substituent selected from the groupconsisting of C₁₋₆alkyl, hydroxyC₁₋₆alkyl and R⁰; (ix) 6-memberedheterocycloalkyl comprising, as ring members, 1 to 2 heteroatomsindependently selected from the group consisting of N and O, and whereinthe 6-membered heterocycloalkyl is unsubstituted or substituted byC₁₋₆alkyl; (x) phenyl that is unsubstituted or substituted by halogen;and (b) C₃₋₆cycloalkyl that is unsubstituted or substituted by 1 to 2substituents independently selected from C₁₋₆haloalkyl, R⁰,C₁₋₆alkylamino, —C(O)R⁰, C₁₋₆alkyl that is unsubstituted or substitutedby —R⁰ or —C(O)R⁰.
 322. The method of claim 318, wherein R² is selectedfrom the group consisting of: n-propyl, isopropyl, t-butyl,


323. The method of claim 322, wherein ring A is: (a) selected from thegroup consisting of

which are each unsubstituted or substituted by a substituent selectedfrom the group consisting of halogen, cyano, C₁₋₆alkyl, C₁₋₆haloalkyl,and —NH₂; or (b) is

 which is unsubstituted or substituted by C₁₋₆alkyl.
 324. The method ofclaim 317, wherein the LATS inhibitor is a compound of Formula II:


325. The method of claim 324, wherein ring A is: (a) selected from thegroup consisting of

which are each unsubstituted or substituted by a substituent selectedfrom the group consisting of halogen, cyano, C₁₋₆alkyl, C₁₋₆haloalkyl,and —NH₂; or (b) is

 which is unsubstituted or substituted by C₁₋₆alkyl.
 326. The method ofclaim 324, wherein ring A is


327. The method of claim 324, wherein R² is selected from the groupconsisting of: n-propyl, isopropyl, t-butyl,


328. The method of claim 327, wherein ring A is: (a) selected from thegroup consisting of

which are each unsubstituted or substituted by a substituent selectedfrom the group consisting of halogen, cyano, C₁₋₆alkyl, C₁₋₆haloalkyl,and —NH₂; or (b) is

 which is unsubstituted or substituted by C₁₋₆alkyl.
 329. The method ofclaim 327, wherein ring A is


330. The method of claim 317, wherein the LATS inhibitor of Formula A1or salt thereof is selected from:N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N,N-diethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-(1,1,1-trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-butyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)ethan-1-ol;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)propan-2-ol;N-ethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-cyclohexylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methyloxetan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;N-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methyl-4-phenylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopropyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methanesulfonyl-2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propoxy)aceticacid;(1R,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-(1-methanesulfonyl-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(2S)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-[(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)amino]aceticacid;(2R)-3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; methyl2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoate;(1S,2S)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentan-1-ol;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid;2-(2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethoxy)ethan-1-ol;2-(hydroxymethyl)-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propane-1,3-diol;3-methyl-3-(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanamido)butanoicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-3-phenylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-{[4-(dimethylamino)oxan-4-yl]methyl}-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-(2-methanesulfonylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(adamantan-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propanamide;4,4,4-trifluoro-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoicacid;N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]propane-2-sulfonamide;2-(pyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(pyridin-4-yl)-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4,4,4-trifluoro-2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopentyl)methanol;N-(3-methoxycyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1R,2R)-1-N,2-N-dimethyl-1-N-[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]cyclohexane-1,2-diamine;methyl(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;ethyl1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylate;1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;(1s,3s)-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutane-1-carboxylicacid;2-(pyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbutan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(pyridin-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propan-1-ol;N-(butan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylbut-3-yn-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(1r,3s)-3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutan-1-ol;2,3-dimethyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-2-ol;2-(pyridin-4-yl)-N-(2,4,4-trimethylpentan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(pentan-3-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(tert-butoxy)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4,4,4-trifluoro-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-pentyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butan-1-ol;N-[1-(1H-indol-3-yl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[1-(4-fluorophenyl)-2-methylpropan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-phenylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-[2-(4-fluorophenyl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3,3,3-trifluoro-2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanoicacid; 2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}ethan-1-ol;N-methyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;1-({[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}methyl)cyclopentan-1-ol;N,N-dimethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(2-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-methoxyphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-phenyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(3-methylphenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;6-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}hexanoic acid;N-(3-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-fluorophenyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanoic acid;N-(1-phenylethyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butylN-(2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propyl)carbamate;(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;methyl2-(1-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)acetate;N-(2-methylpropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butanenitrile;N-(6-aminohexyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(4-aminobutyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-2-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}propanenitrile;N-[2-methyl-1-(2-methylpiperidin-1-yl)propan-2-yl]-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine;N-(1-amino-2-methylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-cyclopentyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-[4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl]pyridin-2-amine;2-[1-(benzenesulfonyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-N-tert-butylpyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(3-methylpyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)pentan-2-ol;N-ethyl-2-(3-fluoropyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-methyl-1-[2-methyl-2-({2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-yl}amino)propoxy]propan-2-ol;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-ethyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(1-methoxy-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;2-(3-chloropyridin-4-yl)-N-cyclopentylpyrido[3,4-d]pyrimidin-4-amine;1-(2-{[2-(3-chloropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2-methylpropoxy)-2-methylpropan-2-ol;N-methyl-2-(3-methylpyridin-4-yl)-N-(propan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-(4-methanesulfonyl-2-methylbutan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[3-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-[2-chloro-5-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-[3-(1H-1,2,3,4-tetrazol-5-yl)propyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-(11,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-fluoropyridin-4-yl)-N-methyl-N-[(2R)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridin-2-amine;2-(3-chloropyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-{2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(1H-indazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(3,5-dimethyl-1H-pyrazol-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1,1,1-trifluoro-2-methylpropan-2-yl)-2-[3-(trifluoromethyl)-1H-pyrazol-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(4-hydroxy-2,4-dimethylpentan-2-yl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-3-carbonitrile;2-(3,5-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(2,3-difluoropyridin-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-thiazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-[2-(trifluoromethyl)pyridin-4-yl]pyrido[3,4-d]pyrimidin-4-amine;4-{4-[(1-methylcyclopropyl)amino]pyrido[3,4-d]pyrimidin-2-yl}pyridine-2-carbonitrile;N-(1-methylcyclopropyl)-2-(1,2-oxazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(dimethyl-1,2-oxazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[2,3-b]pyridin-3-yl}pyrido[3,4-d]pyrimidin-4-amine;N-propyl-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-(1,1,1-trifluoro-2-methylpropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclobutyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-{[2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}-2,4-dimethylpentan-2-ol;N-propyl-2-{7H-pyrrolo[2,3-d]pyrimidin-5-yl}pyrido[3,4-d]pyrimidin-4-amine;2-(3-chloropyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-cyclopropyl-1H-pyrazol-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-(3-methylpyridin-4-yl)-N-propylpyrido[3,4-d]pyrimidin-4-amine;2-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}-N-propylpyrido[3,4-d]pyrimidin-4-amine;2,4-dimethyl-4-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}pentan-2-ol;N-[(1R)-1-phenylethyl]-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(5-methyl-1H-pyrazol-4-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[(1R)-1-phenylethyl]pyrido[3,4-d]pyrimidin-4-amine;N-methyl-N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(pyridazin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1,3-oxazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-imidazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-{1H-pyrrolo[3,2-b]pyridin-1-yl}pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-1,2,3-triazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1,2-oxazol-5-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(2H-1,2,3,4-tetrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-(1-methylcyclobutyl)pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1-methyl-1H-pyrazol-5-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;2-(1H-pyrazol-4-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(trifluoromethyl)cyclopropyl]pyrido[3,4-d]pyrimidin-4-amine;2-(3-methyl-1H-pyrazol-4-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclobutyl)methanol;(1-{[2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}cyclopropyl)methanol;2-(1-methyl-1H-pyrazol-5-yl)-N-[1-(pyridin-4-yl)ethyl]pyrido[3,4-d]pyrimidin-4-amine;N-(1-methylcyclopropyl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-ethyl-1H-pyrazol-4-yl)-N-(2-methylpropyl)pyrido[3,4-d]pyrimidin-4-amine;2-(1-methyl-1H-pyrazol-4-yl)-N-(1-methylcyclopropyl)pyrido[3,4-d]pyrimidin-4-amine;N-(1-amino-2-methylpropan-2-yl)-2-(1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;8-methyl-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-tert-butyl-5-chloro-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;5-chloro-N-(1-methylcyclopropyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;2-(2-{[4-(tert-butylamino)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-5-yl]amino}ethoxy)ethan-1-ol;N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[2-methyl-1-(propan-2-yloxy)propan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2S)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-[(2R)-butan-2-yl]-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methoxy-2-methylpropan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butan-1-ol;N-tert-butyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2,2-dimethyl-1-[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]piperidin-4-ol;2,4-dimethyl-4-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}pentan-2-ol;N-cyclopentyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;dimethyl(3-methyl-3-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}butyl)amine;N,N-diethyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2-methyl-1-(2-methyl-2-{[2-(pyridin-4-yl)-1,7-naphthyridin-4-yl]amino}propoxy)propan-2-ol;N-propyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(3-methyl-1H-pyrazol-4-yl)-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyrimidin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N-tert-butyl-2-{1H-pyrrolo[2,3-b]pyridin-4-yl}-1,7-naphthyridin-4-amine;N-tert-butyl-2-(pyridazin-4-yl)-1,7-naphthyridin-4-amine;2-(2-aminopyridin-4-yl)-N-tert-butyl-1,7-naphthyridin-4-amine;N,N-diethyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;(3-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-3-methylbutyl)dimethylamine;2-(3-fluoropyridin-4-yl)-N-methyl-N-(propan-2-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-4-(piperidin-1-yl)-1,7-naphthyridine;2-(3-fluoropyridin-4-yl)-4-(morpholin-4-yl)-1,7-naphthyridine;N-tert-butyl-2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-amine;2-(3-fluoropyridin-4-yl)-N-(2-methylbutan-2-yl)-1,7-naphthyridin-4-amine;2-{[2-(3-fluoropyridin-4-yl)-1,7-naphthyridin-4-yl]amino}-2-methylpropan-1-ol;1-[2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-yl]-2,2-dimethylpiperidin-4-ol;2-(3-fluoropyridin-4-yl)-N-[2-methyl-1-(morpholin-4-yl)propan-2-yl]-1,7-naphthyridin-4-amine;N-tert-butyl-2-(3-chloropyridin-4-yl)-1,7-naphthyridin-4-amine;N-((1R,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(S)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; N-((1S,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;(R)—N-(sec-butyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; N-((1S,2S)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;N-((1R,2R)-2-methylcyclopentyl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine;4-(4-methylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;4-(piperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;4-(2-methylpiperidin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclobutyl)-1,7-naphthyridin-4-amine;2-methyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine;N-(oxetan-3-yl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methylcyclopropyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;4-(3,3-dimethylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2,2-dimethyl-4-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)morpholine;N-(1-methylcyclobutyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;2,2-dimethyl-N1-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine;N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,2-diamine;4-(2-methylpiperazin-1-yl)-2-(pyridin-4-yl)-1,7-naphthyridine;2-methyl-N¹-(2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)propane-1,3-diamine;(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)-1,7-naphthyridine;N-(tert-butyl)-N-methyl-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine;N-(1-methylcyclobutyl)-2-(pyrimidin-4-yl)-1,7-naphthyridin-4-amine;N¹,N¹,3-trimethyl-N³-(2-(pyrimidin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N¹,N¹,3-trimethyl-N³-(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2-methyl-1-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propan-2-yl)carbamate;tert-butyl(2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)ethyl)carbamate;2-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)ethane-1,2-diamine;N,N,2-trimethyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;N¹,3-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;tert-butyl(2,2-dimethyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propyl)carbamate;2,2-dimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butanamide;(R)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;2,3-dimethyl-N²-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-2,3-diamine;(S)-2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;ethyl2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanoate;N¹N1,2,2-tetramethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;4-(4-(tert-butylamino)pyrido[3,4-d]pyrimidin-2-yl)-1,2,5-oxadiazol-3-amine;N²,N²,2-trimethyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,2-diamine;2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)propanamide;(S)-1,1,1-trifluoro-2-methyl-3-((2-(pyridin-4-yl)-1,7-naphthyridin-4-yl)amino)propan-2-ol;2-(3-chloropyridin-4-yl)-N,N-diethyl-1,7-naphthyridin-4-amine;N-propyl-2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine;tert-butyl(3-methyl-3-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)amino)butyl)carbamate;N¹,N¹,N³,2,2-pentamethyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)propane-1,3-diamine;N¹,N¹-diethyl-3-methyl-N³-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N³-(2-(2-fluoropyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N³-(2-(3,5-dimethyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N¹,N¹,3-trimethylbutane-1,3-diamine;N¹,N¹,3-trimethyl-N³-(2-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine;N³-(2-(2-aminopyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)-N,N¹,3-trimethylbutane-1,3-diamine;and3-methyl-N¹-(2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine.331. The method of claim 317, wherein the LATS inhibitor or salt thereofis selected from:3-(pyridin-4-yl)-N-(1-(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1-amine;N-(1-methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine;2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine;N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine; andN-methyl-2-(pyridin-4-yl)-N-[(2S)-1,1,1-trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.332. The method of claim 317, wherein the LATS inhibitor isN-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine, or a saltthereof.
 333. The method of claim 316, wherein the ocular cells comprisecorneal endothelial cells or limbal stem cells.
 334. The method of claim323, wherein the ocular cells comprise corneal endothelial cells orlimbal stem cells.
 335. The method of claim 331, wherein the ocularcells comprise corneal endothelial cells or limbal stem cells.
 336. Themethod of claim 335, wherein the seeding population of cells comprisesgreater than 20% corneal endothelial cells or greater than 20% limbalstem cells.
 337. The method of claim 316, wherein one or more of saidcells comprises a non-naturally occurring insertion or deletion of oneor more nucleic acid residues of a gene associated with facilitating ahost vs graft immune response, wherein the insertion and/or deletionresults in reduced or eliminated expression or function of said gene.338. The method of claim 337, wherein said gene is selected from: B2M,HLA-A, HLA-B and HLA-C.
 339. The method of claim 337, wherein thenon-naturally occurring insertion and/or deletion has been introducedinto the one or more cells with a gene editing system selected from: aCRISPR gene editing system, a TALEN gene editing system, a zinc fingernuclease gene editing system, a meganuclease gene editing system, AAVvector driven homologous recombination and lentiviral vectors-basedgenome editing technologies.
 340. The method of claim 337, wherein thegene editing system is a CRISPR gene editing system, and the gene isB2M.
 341. The method of claim 340, wherein the seeding cell populationcomprises at least 21% of B2M-negative cells.
 342. The method of claim323, wherein one or more of said cells comprises a non-naturallyoccurring insertion and/or deletion of one or more nucleic acid residuesof the B2M gene, wherein the insertion and/or deletion results inreduced or eliminated expression or function of said B2M gene, andwherein the non-naturally occurring insertion and/or deletion has beenintroduced into the one or more cells with a CRISPR gene editing system.343. The method of claim 331, wherein one or more of said cellscomprises a non-naturally occurring insertion and/or deletion of one ormore nucleic acid residues of the B2M gene, wherein the insertion and/ordeletion results in reduced or eliminated expression or function of saidB2M gene, and wherein the non-naturally occurring insertion and/ordeletion has been introduced into the one or more cells with a CRISPRgene editing system.
 344. A cell population produced by the method ofclaim
 316. 345. A cell population produced by the method of claim 323.346. A cell population produced by the method of claim
 331. 347. A cellpopulation produced by the method of claim
 343. 348. A pharmaceuticalcomposition comprising the cell population of claim 344 and at apharmaceutically acceptable excipient.
 349. A pharmaceutical compositioncomprising the cell population of claim 347 and at a pharmaceuticallyacceptable excipient.